Methods of treating mitochondrial disorders

ABSTRACT

Provided herein are methods for treating a disease or disorder associated with mitochondrial dysfunction through ex vivo introduction of a nucleic acid molecule into hematopoietic stem and progenitor cells (HSPCs) followed by transplantation of the HSPCs into a subject in need of treatment. The nucleic acid molecule may include a functional human frataxin (hFXN) or may include a gene editing system that when transfected into the cells removes a trinucleotide extension mutation of endogenous hFXN.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of priority under 35 U.S.C. § 119(e)of U.S. Ser. No. 62/312,105, filed Mar. 23, 2016, the entire content ofwhich is incorporated herein by reference.

GRANT INFORMATION

This invention was made with government support under Grant No.R21NS090066 awarded by the National Institutes of Health. The UnitedStates government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 13, 2017, isnamed 20378-201301_SL.txt and is 23,442 bytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates generally to mitochondrial disease and morespecifically to methods of treating mitochondrial diseases withhematopoietic stem and progenitor cell (HSPC) gene therapy.

Background Information

Mitochondrial disease is a group of disorders caused by dysfunctionalmitochondria, the organelles that are the powerhouse of the cell.Mitochondria are found in every cell of the human body except red bloodcells, and convert the energy of food molecules into the ATP that powersmost cell functions. Mitochondrial diseases are sometimes caused bymutations in the mitochondrial DNA that affect mitochondrial function.Other causes of mitochondrial disease are mutations in genes of thenuclear DNA, whose gene products are imported into the mitochondria(mitochondrial proteins) as well as acquired mitochondrial conditions.Mitochondrial diseases take on unique characteristics both because ofthe way the diseases are often inherited and because mitochondria are socritical to cell function. The subclass of these diseases that haveneuromuscular disease symptoms are often called mitochondrialmyopathies. Symptoms associated with mitochondrial disease typicallyinclude poor growth, loss of muscle coordination, muscle weakness,visual problems, hearing problems, learning disabilities, heart disease,liver disease, kidney disease, gastrointestinal disorders, respiratorydisorders, neurological problems, autonomic dysfunction and dementia.

Mitochondrial diseases/disorders may be caused by mutations, acquired orinherited, in mitochondrial DNA (mtDNA) or in nuclear genes that codefor mitochondrial components. They may also be the result of acquiredmitochondrial dysfunction due to adverse effects of drugs, infections,or other environmental causes.

One of the most common inherited autosomal recessive diseases associatedwith reduced expression of the nuclear-encoded mitochondrial protein,frataxin, is Friedreich's ataxia (FRDA) which affects people at an earlyage. Point mutations have also been described resulting in truncated ordysfunctional frataxin. FRDA is characterized by ataxia, areflexia,sensory loss, muscle weakness, and cardiomyopathy. Symptoms typicallybegin between 5 to 15 years of age and patients will be in a wheelchairwithin 10-15 years of onset.

FRDA is caused, in 98% of all cases, by a genetic mutation resulting inexpansion of GAA repeats in the first intron of the frataxin gene (FXN).In healthy individuals the alleles may contain up to about 40 GAArepeats, whereas expanded alleles in FRDA patients can consist of 90 to1700 repeats (SEQ ID NO: 12) (see FIG. 1B). The GAA repeat expansionleads to reduced expression of frataxin, a highly conservedmitochondrial protein mainly expressed in mitochondria-rich tissuesincluding the nervous system, muscle, and heart. Also, carriers(heterozygous for the expanded allele) show ˜50% reduction of frataxinmRNA and protein levels compared to normal expression, although they donot show any symptoms. While its function is not fully elucidated,frataxin is an iron binding protein participating in Fe-S clusterassembly and in its absence, iron accumulates within mitochondrialeading to defective iron-mediated biosynthetic processes and increasedoxidative stress.

Expanded GAA repeats form an intramolecular triple-helix (triplex),so-called H-DNA, in supercoiled plasmids isolated from E. coli. Severalmodels representing the triplex structures formed at expanded GAArepeats are proposed, and direct evidence for a pyrimidine motif H-DNAstructure at pathological GAA expansions in vitro has recently beenprovided. Also, formation of a higher order structure named “sticky DNA”has been observed in frataxin GAA repeats-containing plasmids using gelelectrophoresis and atomic force microscopy. The molecular structure ofsticky DNA is not resolved; however, current evidence demonstrates thatsticky DNA forms as one long intramolecular triplex structure or by theassociation of two triplexes.

The observed effects on DNA replication and transcription are dependenton the length and orientation of the GAA repeats in plasmids, whichcorrelate with formation of the specific DNA structure (H-DNA). Finally,the GAA repeats are associated with a pattern of DNA methylation andhistone acetylation in the adjacent regions and the formation ofsilenced chromatin. The presence of H-DNA and higher order structureswithin the GAA repeats is believed to recruit chromatin-remodelingprotein complexes that maintain a close chromatin structure leading todown-regulation of frataxin gene transcription.

Numerous data have demonstrated that analysis of GAA repeats constitutean essential part in the diagnosis of FRDA along with clinicaldiagnosis. Molecular genetic tests are also performed to identifycarriers and in prenatal testing. Current FA diagnostic methods involvepolymerase chain reaction (PCR) analysis and Southern blottingtechnique. The PCR test is performed by amplification of the GAArepeat-containing DNA region in the frataxin gene. The different PCRreactions that have been employed to map GAA repeat expansions areclassical PCR, long-range PCR or triplet-primed PCR (TP-PCR). In allcases, the size of the PCR fragment is analyzed using agarose-gelelectrophoresis and DNA sequencing. In most cases, both PCR and Southernblot are combined to complement the results. Problems encountered duringamplification of medium- and long-sized GAA repeats (i.e., number ofrepeats >200) using PCR have been reported. The repetitive nature of theexpanded sequence and its ability to adopt H-DNA and higher order DNAstructures are the two main factors causing polymerase pausing leadingto false results.

To date, there are no known cures or preventative measures for suchmitochondrial diseases, with current therapies being directed totreating the associated symptoms. Thus, there is a need in the art foralternative or improved methods for treating mitochondrialdiseases/disorders.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention provides a method of treatinga mitochondrial disease or disorder in a subject. The method includesintroducing ex vivo a functional human frataxin (hFXN) intohematopoietic stem and progenitor cells (HSPCs) of the subject, andtransplanting the HSPCs into the subject, thereby treating themitochondrial disease or disorder. The step of introducing may includecontacting a vector comprising a polynucleotide encoding hFXN and a FXNpromoter (or other regulatory sequence that is operable with thepolynucleotide and in the cell) with the HSPCs and allowing expressionof hFXN. In various embodiments, the mitochondrial disease or disorderis selected from the group consisting of Friedreich's ataxia (FRDA),diabetes, Leigh syndrome, Leber's hereditary optic neuropathy,myoneurogenic gastrointestinal encephalopathy, and cancer. The subjectmay be a mammal, such as a human. In various embodiments, the vector isa self-inactivating (SIN)-lentivirus vector, such as pCCL-FRDAp-FXN. Invarious embodiments, expression of hFXN corrects neurologic, cardiac andmuscular complications within about 6-12 months post-transplantation. Inanother aspect, the hFXN polynucleotide is introduced into HSPCs in vivoin a subject.

In another aspect, the present invention provides a method of treating amitochondrial disease or disorder in a subject comprising contactingcells expressing hFXN from the subject with a vector encoding a geneediting system that when transfected into the cells removes atrinucleotide extension mutation of endogenous hFXN, thereby treatingthe mitochondrial disease or disorder. In various embodiments, the geneediting system is selected from the group consisting of CRISPR/Cas, zincfinger nucleases, and transcription activator-life effector nucleases.The step of contacting may include obtaining a sample of cells from thesubject, transfecting or transducing the gene editing system into thesample of cells to create gene-corrected cells, and thereafter,transplanting the gene-corrected cells into the subject. The sample ofcells may be any cells expressing hFXN, such as blood cells and HSPCsfrom the subject.

In another aspect, the present invention provides an expression cassettecomprising a promoter or regulatory sequence functionally linked to apolynucleotide encoding hFXN. Also provided are a vector, such as aself-inactivating (SIN)-lentivirus vector, that includes a regulatorysequence such as a promoter functionally linked to a polynucleotideencoding hFXN.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are graphical and pictorial diagrams showing that systemictransplantation of WT HSPCs prevents sensory neuron degeneration andneurobehavioral deficits in YG8R mice. FIG. 1A shows the results of WT(n=16), YG8R control (n=4), YG8R/YG8R HSPCs (n=5) and YG8R/WT HSPCs(n=13) mice at both 5 and 9 months of age. Locomotor activity was testedusing an open field, coordination using a rotarod, gait using anautomated gait analysis system and muscle strength using forelimb gripstrength. Data are expressed as means±sem; *P<0.05, **P<0.005,***P<0.0005; NS statistically non-significant. For statisticalcomparison of three experimental groups, a mixed analysis of variance(ANOVA) with age of testing as a within-subjects variable was usedfollowed by independent sample t-test. FIG. 1B is a representationshowing intron 1 of an unaffected (top) frataxin gene (FXN) and intron 1of FRDA (bottom) FXN, and discloses SEQ ID NOs: 15-16, respectively, inorder of appearance. FIG. 1C shows Niss1-stained sections of lumbar DRG(L5) from representative 9-month-old WT (n=15), YG8R control (n=4),YG8R/YG8R HSPCs (n=4) and YG8R/WT HSPCs (n=11) mice. DRGs of YG8Rcontrols exhibit large vacuoles (arrows). Scale bars, 100 μm. Graph onthe right depicts total vacuole area per DRG area; data are expressed asmeans±sem; **P<0.005; ***P<0.0005. NS, statistically non-significant.FIG. 1D shows representative confocal images from a WT GFP⁺HSPC-transplanted YG8R mouse 7 months post-transplantation stained withanti-GFP and anti-NeuN. Left: Image of a lumbar (L5) DRG illustratesengraftment of GFP⁺ HSPC-derived cells throughout DRG. Scale bar, 100μm. Magnified image (below) demonstrates frequent close association ofHSPC-derived cells with DRG neurons. Scale bar, 20 μm. Right: Images ofcervical, thoracic and lumbar spinal cord show abundant HSPC engraftmentthroughout spinal cord gray and white matter at all levels. Scale bars,250 μm. FIG. 1E shows confocal images of DRG and spinal cord sections ofa GFP⁺ HSPC-treated YG8R mouse. Engrafted cells (GFP) are closelyassociated with neurons (NeuN), and co-localization with Iba1 marker;Scale bars: 30 μm.

FIGS. 2A-2E are graphical and pictorial diagrams showing thattransplanted HSPCs engraft throughout the brain and preventfrataxin-deficiency toxicity. FIG. 2A shows representative transversesections of the brain of a WT GFP⁺ HSPC-transplanted YG8R mouse 7 monthspost-transplantation labeled with anti-GFP and anti-NeuN. Scale bar, 1mm. Magnified picture #1 of the brain shows that GFP⁺ HSPC-derived cellsare observed in periventricular regions including the corpus callosum(cc), lateral septal nuclei (LS), caudate putamen (CP), anteriorcingulate area (ACA), and the somatosensory cortex (M1, S2). VL, lateralventricle. Scale bar, 150 μm. Magnified picture #2 of ventral striatumof the brain shows that the engrafted GFP⁺ HSPCs are present in regionsof the ventral striatum including the anterior commissure (aco), nucleusaccumbens (ACB), and lateral septal nuclei (LS). CP, caudate putamen.Scale bar, 150 μm. Magnified picture #3 shows that GFP⁺ HSPC-derivedcells are observed in the ventral pallidum (PAL) and the ventralstriatum, including the islands of Calleja (isl) and the olfactorytubercle (OT). Scale bar, 150 μm. GFP⁺ HSPCs were also detected throughgray and white matter of the brainstem and cerebellum. Scale bar, 500μm. Insets depict engraftment within the dentate nucleus (DN) of thecerebellum and the spinal trigeminal nucleus (Sp) of the brainstem.Scale bar, 50 μm. FIG. 2B shows confocal image of brain labeled withanti-GFP, anti-Iba1 and anti-NeuN. Most of the bone marrow-derived GFP⁺cells co-localize with the microglial marker Iba1. Scale bar, 30 μm.FIG. 2C shows quantification of murine frataxin mRNA expression incerebellum from WT (n=14), YG8R (n=8) and YG8R/HSPCs (n=13) mice. Dataare represented as fold change relative to WT normalized to GAPDH. Dataare expressed as means±sem; **P<0.005, ***P<0.0005. FIG. 2D shows theresults of a representative Western blot showing the level of oxidationin cerebrum of one WT, one YG8R, one YG8R/YG8R HSPCs and one YG8R/WTHSPCs mouse with (+) or without (−) derivatization reagent. Oxyblotanalysis detected significantly higher level of oxidized proteins incerebrum of 9-month-old YG8R (n=4) and YG8R/YG8R HSPCs (n=4) compared toWT (n=6) and YG8R/HSPCs (n=6) mice. Data are expressed as means±sem;*P<0.05, NS statistically non-significant. FIG. 2E shows scatter plotsof mitochondrial gene changes in cerebrum from WT animals (n=3) comparedto YG8R (n=3) (left scatter plot) or YG8R/WT HSPCs mice (n=3) (rightscatter plot). The center line represents the cipher, and upregulatedand downregulated genes are noted by dots, respectively. mRNA changesthat are significantly different between groups are represented on aseparate bar graph. Data are expressed as means±sem; *P<0.05, **P<0.005,***P<0.0005, NS statistically non significant as compared to WT.

FIGS. 3A-3H are pictorial and graphical diagrams showing transplantedHSPCs engraft abundantly in heart and muscle. FIG. 3A shows the resultsof a representative Western blot showing level of oxidation in skeletalmuscle of one WT, one YG8R and one YG8R/HSPCs mouse with (+) or without(−) derivatization reagent. Oxyblot analysis detects high level ofprotein oxidation only in skeletal muscle of 9-month-old YG8R controls(YG8R, n=4 and YG8R/YG8R HSPCs, n=5) compared to WT (n=16) andYG8R/HSPCs (n=13) mice. Error bars indicate SEM. *p<0.05, NSstatistically non-significant. FIG. 3B shows quantification of lactateand pyruvate by mass-spectrometry in muscle tissues from WT (n=6), YG8R(n=3) and YG8R/WT HSPCs (n=5) mice. The lactate/pyruvate ratio issignificantly increased in the YG8R mice compared to WT while comparablein YG8R/WT HSPCs animals. Error bars indicate sem; *P<0.05, ***P<0.0005,NS statistically non-significant. FIG. 3C shows representative Perl'sstaining of heart sections from 18 month old WT, YG8R control andYG8R/WT HSPCs. Characteristic staining indicates iron deposition. Scalebars, 50 μm and 15 μm (zoom). The associated bar graph shows ironquantification in heart sections from WT (n=4), YG8R controls (YG8R(n=2), YG8R/YG8R HSPCs (n=2)), and YG8R/WT HSPCs (n=3). Error barsindicate sem; *P<0.05, NS statistically non-significant. FIGS. 3D-3Eshow quantification of murine frataxin mRNA expression in heart (FIG.3D) and skeletal muscle (FIG. 3E) from WT (n=12), YG8R (n=7) andYG8R/HSPCs (n=11) mice. Data are represented as fold change relative toWT normalized to GAPDH, error bars indicate sem; *P<0.05, **P<0.005,***P<0.0005, NS statistically non-significant. FIG. 3F shows an image ofa heart section from WT HSPCs transplanted YG8R mouse 7 monthspost-transplantation stained with anti-GFP, the cardiomyocyte markeranti-α-actinin and DAPI. GFP⁺ cells are found in all the cardiac tissuewith a highest expression in the valve suggesting that HSPCs derivedcells are entering the heart by the blood flow. Scale bar, 150 μm.Magnified pictures of the heart show high level of engraftment in theleft ventricle (bottom) and in the base of the aorta (top). Scale bars,50 μm. FIG. 3G shows skeletal muscle section from WT HSPCs transplantedYG8R mouse 7 months post-transplantation stained with anti-GFP,filamentous actin dye Phalloidin and DAPI. GFP⁺ cells are engraftedhomogenously in the tissue. Scale bar, 150 μm. Magnified picture of theskeletal muscle (on the left) shows that GFP⁺ cells are localizedinterstitially between muscle fibers. Scale bar, 50 μm. FIG. 3H showsquantification of murine MuRF-1, Atrogin-1 and myostatin mRNA expressionin skeletal muscle from WT (n=5), YG8R (n=5) and YG8R/HSPCs (n=5) mice.Data are represented as fold change relative to WT normalized to GAPDH,error bars indicate sem; *P<0.05, NS statistically non-significant.

FIGS. 4A-4F are pictorial and graphical diagrams showing thatHSPC-derived cells deliver frataxin-bearing mitochondria to the diseasedcells in vitro and in vivo. FIGS. 4A and 4B show representative framesfrom confocal imaging movies of YG8R-derived fibroblasts (F) co-culturedwith primary macrophages (M) isolated from a DsRed Cox8-GFP transgenicmouse (FIG. 4A) or with IC21 macrophages transduced with a LV-hFXN-GFPand stained with a red MitoTracker (FIG. 4B). Scale bar, 10 μm. FIG. 4Cshows a representative confocal image of brain sections from an YG8Rmouse transplanted with DsRed⁺ HSPCs (control) and brain and spinal cordsections from an YG8R mouse transplanted with DsRed⁺/Cox8-GFP⁺ HSPCs at7 months post-transplantation labelled with an anti-NeuN antibody. Inaddition to the DsRed-derived bone marrow cells, cox8-GFP are observedin host neurons in brain and spinal cord (arrows). For DRG, heart andmuscle, see FIGS. 7A and 7B. Scale bars, 10 μm. FIG. 4D showsrepresentative confocal images of spinal cord section from an YG8R mousetransplanted with DsRed⁺/Cox8-GFP⁺ HSPCs at 7 monthspost-transplantation labelled with an anti-NeuN antibody showingcox8-GFP within the branch extension of the DsRed⁺ microglial cell(arrows). Scale bar, 5 μm. FIG. 4E shows quantification of neuronscontaining cox8-GFP in the cervical spinal cord gray matter of YG8R micetransplanted with DsRed⁺/Cox8-GFP⁺ HSPCs at 7 monthspost-transplantation (for description of the automatic unbiasedquantification method see FIG. 8). FIG. 4F shows representative confocalimages of brain and spinal cord sections from an YG8R mouse transplantedwith DsRed⁺ HSPCs transduced with LV-hFXN-GFP at 7 monthspost-transplantation and stained with anti-mcherry and anti-NeuNantibodies. In addition to the DsRed-derived bone marrow cells,frataxin-GFP are observed in host neurons. Scale bar, 10 μm.

FIG. 5 is a pictorial diagram showing that HSPCs engraft in theperipheral nerve in YG8R mice. Confocal images of sciatic nerve from WTGFP⁺ HSPC-transplanted YG8R mice labeled with anti-GFP, and with aneurofilament marker, anti-NF200, and a myelin basic protein marker,anti-MBP. Scale bars: 100 μm (left), 10 μm (inset).

FIGS. 6A-6F are pictorial diagrams showing that HSPCs differentiate intomacrophages in DRG and microglia in the spinal cord and brain. FIGS. 6Aand 6B show confocal images of DRG, spinal cord and brain sections fromWT GFP⁺ HSPC-transplanted YG8R mice labeled with anti-GFP, anti-CD68(FIG. 6A), anti-MHCII (FIG. 6B), anti-NeuN (FIG. 6A), and DAPI. Scalebars, 30 μm. FIGS. 6C and 6D show transverse spinal cord (FIG. 6C) andbrain (FIG. 6D) section from WT GFP⁺ HSPC-transplanted YG8R mouselabeled with anti-MHCII. Scale bars, 100 μm (FIG. 6C) and 300 μm (FIG.6D). FIG. 6E shows a confocal image of brain section from WT GFP⁺HSPC-transplanted YG8R mouse labeled with anti-vwf. Scale bar, 50 μm.FIG. 6F shows a confocal image of choroid plexus from WT DsRed⁺HSPC-transplanted YG8R mouse labeled with anti-RFP and anti-Iba1. Scalebar, 100 μm.

FIGS. 7A and 7B are pictorial diagrams showing that HSPCs differentiateinto macrophages in heart and muscle. Confocal images of heart andskeletal muscle section from YG8R transplanted with WT GFP⁺ HSPCs afterlabeling with anti-GFP, anti-CD68 (FIG. 7A) anti-MHCII (FIG. 7B),Phalloidin and DAPI. Scale bar, 30 μm.

FIG. 8 is a pictorial diagram showing that HSPC-derived macrophagesdeliver mitochondria to neurons in DRG and to myocytes in heart andskeletal muscle. Representative confocal images of DRG, heart andskeletal muscle from an YG8R mouse transplanted with DsRed⁺/Cox8-GFP⁺HSPCs at 7 months post-transplantation stained with anti-NeuN (DRG),anti-α-Actinin (heart) or Palloidin (muscle), and DAPI (heart andmuscle). Scale bars, 10 μm.

FIGS. 9A-9D are pictorial and graphical diagrams showing quantificationof Cox8-GFP transfer from HSPC-derived microglia to neurons. FIG. 9Ashows a representative transverse image of cervical spinal cord graymatter from a YG8R mouse at 7 months following transplantation withCox8-GFP DsRed HSPCs, stained with anti-NeuN. Scale bar, 500 μm. FIG. 9Bshows automatic outline and quantification of neurons by ImageProsoftware. FIG. 9C shows that GFP signal is only counted within thedelineated neurons (arrow) and not outside (star). FIG. 9D shows thepercentage of neurons within the gray matter of the spinal cord thatcontain GFP for three different animals (transplanted) and for onecontrol. The entire gray matter from three experimental animals and onecontrol (three sections per animal) were quantified.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding of complete phenotypiccorrection of mitochondrial disorders occurs after a singletransplantation of wildtype hematopoietic stem and progenitor cells,which differentiated into phagocytic cells in the nervous system, muscleand heart leading to the neuronal and myocyte cross-correction. There isa pressing need to identify effective therapies for mitochondrialdisorders such as FRDA for which there remains no treatment. To date,preclinical studies using stem cells or gene therapy have had limitedsuccess, or have been restricted to assessment of specific tissues.

The present disclosure demonstrates that a self-inactivating(SIN)-lentivirus vector containing the human frataxin (hFXN) cDNA aswell as the optimal promoter can be used to ex vivo gene-correctedpatients' autologous hematopoietic stem and progenitor cells (HSPCs),which can then be re-transplant in the patients to repopulate their bonemarrow, which will be a reservoir of “healthy” cells for the rest of thelife of the patients. These cells mobilize and integrate into thediseased tissues (brain, muscle, heart), and will lead to their rescue.While autologous HSPCs are used in the illustrative examples herein, oneof skill in the art would recognize that other HSPCs would be useful aswell (e.g., allogeneic).

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

The term “comprising,” which is used interchangeably with “including,”“containing,” or “characterized by,” is inclusive or open-ended languageand does not exclude additional, unrecited elements or method steps. Thephrase “consisting of” excludes any element, step, or ingredient notspecified in the claim. The phrase “consisting essentially of” limitsthe scope of a claim to the specified materials or steps and those thatdo not materially affect the basic and novel characteristics of theclaimed invention. The present disclosure contemplates embodiments ofthe invention compositions and methods corresponding to the scope ofeach of these phrases. Thus, a composition or method comprising recitedelements or steps contemplates particular embodiments in which thecomposition or method consists essentially of or consists of thoseelements or steps.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

The term “subject” or “host organism,” as used herein, refers to anyindividual or patient to which the subject methods are performed.Generally the subject is human, although as will be appreciated by thosein the art, the subject may be an animal. Thus other animals, includingmammals such as rodents (including mice, rats, hamsters and guineapigs), cats, dogs, rabbits, farm animals including cows, horses, goats,sheep, pigs, etc., and primates (including monkeys, chimpanzees,orangutans and gorillas) are included within the definition of subject.

The term “therapeutically effective amount” or “effective amount” meansthe amount of a compound or pharmaceutical composition that will elicitthe biological or medical response of a tissue, system, animal or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician. Thus, the term “therapeutically effective amount” isused herein to denote any amount of a formulation that causes asubstantial improvement in a disease condition when applied to theaffected areas repeatedly over a period of time. The amount will varywith the condition being treated, the stage of advancement of thecondition, and the type and concentration of formulation applied.Appropriate amounts in any given instance will be readily apparent tothose skilled in the art or capable of determination by routineexperimentation.

A “therapeutic effect,” as used herein, encompasses a therapeuticbenefit and/or a prophylactic benefit as described herein.

The terms “administration” or “administering” are defined to include anact of providing a compound or pharmaceutical composition of theinvention to a subject in need of treatment. The phrases “parenteraladministration” and “administered parenterally” as used herein meansmodes of administration other than enteral and topical administration,usually orally or by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid,intraspinal and infrasternal injection and infusion. The phrases“systemic administration,” “administered systemically,” “peripheraladministration” and “administered peripherally” as used herein mean theadministration of a compound, drug or other material other than directlyinto the central nervous system, such that it enters the subject'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration.

If a viral vector specific for the cell type is not available, thevector can be modified to express a receptor (or ligand) specific for aligand (or receptor) expressed on the target cell, or can beencapsulated within a liposome, which also can be modified to includesuch a ligand (or receptor). A peptide agent can be introduced into acell by various methods, including, for example, by engineering thepeptide to contain a protein transduction domain such as the humanimmunodeficiency virus TAT protein transduction domain, which canfacilitate translocation of the peptide into the cell. In addition,there are a variety of biomaterial-based technologies such as nano-cagesand pharmacological delivery wafers (such as used in brain cancerchemotherapeutics) which may also be modified to accommodate thistechnology.

The viral vectors most commonly assessed for gene transfer are based onDNA-based adenoviruses (Ads) and adeno-associated viruses (AAVs) andRNA-based retroviruses and lentiviruses. Lentivirus vectors have beenmost commonly used to achieve chromosomal integration.

As used herein, the terms “reduce” and “inhibit” are used togetherbecause it is recognized that, in some cases, a decrease can be reducedbelow the level of detection of a particular assay. As such, it may notalways be clear whether the expression level or activity is “reduced”below a level of detection of an assay, or is completely “inhibited.”Nevertheless, it will be clearly determinable, following a treatmentaccording to the present methods.

As used herein, “treatment” or “treating” means to administer acomposition to a subject or a system with an undesired condition. Thecondition can include a disease or disorder. “Prevention” or“preventing” means to administer a composition to a subject or a systemat risk for the condition. The condition can include a predisposition toa disease or disorder. The effect of the administration of thecomposition to the subject (either treating and/or preventing) can be,but is not limited to, the cessation of one or more symptoms of thecondition, a reduction or prevention of one or more symptoms of thecondition, a reduction in the severity of the condition, the completeablation of the condition, a stabilization or delay of the developmentor progression of a particular event or characteristic, or minimizationof the chances that a particular event or characteristic will occur.

As used herein, the term “genetic modification” is used to refer to anymanipulation of an organism's genetic material in a way that does notoccur under natural conditions. Methods of performing such manipulationsare known to those of ordinary skill in the art and include, but are notlimited to, techniques that make use of vectors for transforming cellswith a nucleic acid sequence of interest. Included in the definition arevarious forms of gene editing in which DNA is inserted, deleted orreplaced in the genome of a living organism using engineered nucleases,or “molecular scissors.” These nucleases create site-specificdouble-strand breaks (DSBs) at desired locations in the genome. Theinduced double-strand breaks are repaired through nonhomologousend-joining (NHEJ) or homologous recombination (HR), resulting intargeted mutations (i.e., edits).

There are several families of engineered nucleases used in gene editing,for example, but not limited to, meganucleases, zinc finger nucleases(ZFNs), transcription activator-like effector-based nucleases (TALEN),and the CRISPR-Cas system.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is anacronym for DNA loci that contain multiple, short, direct repetitions ofbase sequences. The prokaryotic CRISPR/Cas system has been adapted foruse as gene editing (silencing, enhancing or changing specific genes)for use in eukaryotes (see, for example, Cong, Science,15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21(2012)). By transfecting a cell with elements including a Cas gene andspecifically designed CRISPRs, nucleic acid sequences can be cut andmodified at any desired location. Methods of preparing compositions foruse in genome editing using the CRISPR/Cas systems are described indetail in US Pub. No. 2016/0340661, US Pub. No. 20160340662, US Pub. No.2016/0354487, US Pub. No. 2016/0355796, US Pub. No. 20160355797, and WO2014/018423, which are specifically incorporated by reference herein intheir entireties.

Thus, as used herein, “CRISPR system” refers collectively to transcriptsand other elements involved in the expression of or directing theactivity of CRISPR-associated (“Cas”) genes, including sequencesencoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.,tracrRNA or an active partial tracrRNA), a tracr-mate sequence(encompassing a “direct repeat” and a tracrRNA-processed partial directrepeat in the context of an endogenous CRISPR system), a guide sequence(also referred to as a “spacer”, “guide RNA” or “gRNA” in the context ofan endogenous CRISPR system), or other sequences and transcripts from aCRISPR locus. One or more tracr mate sequences operably linked to aguide sequence (e.g., direct repeat-spacer-direct repeat) can also bereferred to as “pre-crRNA” (pre-CRISPR RNA) before processing or crRNAafter processing by a nuclease.

In some embodiments, a tracrRNA and crRNA are linked and form a chimericcrRNA-tracrRNA hybrid where a mature crRNA is fused to a partialtracrRNA via a synthetic stem loop to mimic the natural crRNA:tracrRNAduplex as described in Cong, Science, 15:339(6121):819-823 (2013) andJinek, et al., Science, 337(6096):816-21 (2012)). A single fusedcrRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA(or single-guide RNA (sgRNA)). Within an sgRNA, the crRNA portion can beidentified as the ‘target sequence’ and the tracrRNA is often referredto as the ‘scaffold’.

There are many resources available for helping practitioners determinesuitable target sites once a desired DNA target sequence is identified.For example, numerous public resources, including a bioinformaticallygenerated list of about 190,000 potential sgRNAs, targeting more than40% of human exons, are available to aid practitioners in selectingtarget sites and designing the associate sgRNA to affect a nick ordouble strand break at the site. See also, crispr.u-psud.fr, a tooldesigned to help scientists find CRISPR targeting sites in a wide rangeof species and generate the appropriate crRNA sequences.

In some embodiments, one or more vectors driving expression of one ormore elements of a CRISPR system are introduced into a target cell suchthat expression of the elements of the CRISPR system direct formation ofa CRISPR complex at one or more target sites. While the specifics can bevaried in different engineered CRISPR systems, the overall methodologyis similar. A practitioner interested in using CRISPR technology totarget a DNA sequence can insert a short DNA fragment containing thetarget sequence into a guide RNA expression plasmid. The sgRNAexpression plasmid contains the target sequence (about 20 nucleotides),a form of the tracrRNA sequence (the scaffold) as well as a suitablepromoter and necessary elements for proper processing in eukaryoticcells. Such vectors are commercially available (see, for example,Addgene). Many of the systems rely on custom, complementary oligos thatare annealed to form a double stranded DNA and then cloned into thesgRNA expression plasmid. Co-expression of the sgRNA and the appropriateCas enzyme from the same or separate plasmids in transfected cellsresults in a single or double strand break (depending of the activity ofthe Cas enzyme) at the desired target site.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymesgenerated by fusing a zinc finger DNA-binding domain to a DNA-cleavagedomain. Zinc finger domains can be engineered to target specific desiredDNA sequences and this enables zinc-finger nucleases to target uniquesequences within complex genomes. By taking advantage of endogenous DNArepair machinery, these reagents can be used to precisely alter thegenomes of higher organisms. The most common cleavage domain is the TypeIIS enzyme Fok1. Fok1 catalyzes double-stranded cleavage of DNA, at 9nucleotides from its recognition site on one strand and 13 nucleotidesfrom its recognition site on the other. See, for example, U.S. Pat. Nos.5,356,802; 5,436,150 and 5,487,994; as well as Li et al. Proc., Natl.Acad. Sci. USA 89 (1992):4275-4279; Li et al. Proc. Natl. Acad. Sci.USA, 90:2764-2768 (1993); Kim et al. Proc. Natl. Acad. Sci. USA.91:883-887 (1994a); Kim et al. J. Biol. Chem. 269:31,978-31,982 (1994b),all of which are incorporated herein by reference. One or more of theseenzymes (or enzymatically functional fragments thereof) can be used as asource of cleavage domains.

Transcription activator-like effector nucleases (TALENs) have an overallarchitecture similar to that of ZFNs, with the main difference beingthat the DNA-binding domain comes from TAL effector proteins,transcription factors from plant pathogenic bacteria. The DNA-bindingdomain of a TALEN is a tandem array of amino acid repeats, each about 34residues long. The repeats are very similar to each other; typicallythey differ principally at two positions (amino acids 12 and 13, calledthe repeat variable diresidue, or RVD). Each RVD specifies preferentialbinding to one of the four possible nucleotides, meaning that each TALENrepeat binds to a single base pair, though the NN RVD is known to bindadenines in addition to guanine. TAL effector DNA binding ismechanistically less well understood than that of zinc-finger proteins,but their seemingly simpler code could prove very beneficial forengineered-nuclease design. TALENs also cleave as dimers, haverelatively long target sequences (the shortest reported so far binds 13nucleotides per monomer) and appear to have less stringent requirementsthan ZFNs for the length of the spacer between binding sites. Monomericand dimeric TALENs can include more than 10, more than 14, more than 20,or more than 24 repeats. Methods of engineering TAL to bind to specificnucleic acids are described in Cermak, et al, Nucl. Acids Res. 1-11(2011); US Published Application No. 2011/0145940, which discloses TALeffectors and methods of using them to modify DNA; Miller et al. NatureBiotechnol 29: 143 (2011) reported making TALENs for site-specificnuclease architecture by linking TAL truncation variants to thecatalytic domain of Fok1 nuclease. The resulting TALENs were shown toinduce gene modification in immortalized human cells. General designprinciples for TALE binding domains can be found in, for example, WO2011/072246. Each of the foregoing references are incorporated herein byreference in their entireties.

The nuclease activity of the genome editing systems described hereincleave target DNA to produce single or double strand breaks in thetarget DNA. Double strand breaks can be repaired by the cell in one oftwo ways: non-homologous end joining, and homology-directed repair. Innon-homologous end joining (NHEJ), the double-strand breaks are repairedby direct ligation of the break ends to one another. As such, no newnucleic acid material is inserted into the site, although some nucleicacid material may be lost, resulting in a deletion. In homology-directedrepair, a donor polynucleotide with homology to the cleaved target DNAsequence is used as a template for repair of the cleaved target DNAsequence, resulting in the transfer of genetic information from a donorpolynucleotide to the target DNA. As such, new nucleic acid material canbe inserted/copied into the site. Therefore, in some embodiments, thegenome editing vector or composition optionally includes a donorpolynucleotide. The modifications of the target DNA due to NHEJ and/orhomology-directed repair can be used to induce gene correction, genereplacement, gene tagging, transgene insertion, nucleotide deletion,gene disruption, gene mutation, etc.

Accordingly, cleavage of DNA by the genome editing vector or compositioncan be used to delete nucleic acid material from a target DNA sequenceby cleaving the target DNA sequence and allowing the cell to repair thesequence in the absence of an exogenously provided donor polynucleotide.Alternatively, if the genome editing composition includes a donorpolynucleotide sequence that includes at least a segment with homologyto the target DNA sequence, the methods can be used to add, i.e., insertor replace, nucleic acid material to a target DNA sequence (e.g., to“knock in” a nucleic acid that encodes for a protein, an siRNA, anmiRNA, etc.), to add a tag (e.g., 6×His (SEQ ID NO: 13), a fluorescentprotein (e.g., a green fluorescent protein; a yellow fluorescentprotein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatorysequence to a gene (e.g., promoter, polyadenylation signal, internalribosome entry sequence (IRES), 2A peptide, start codon, stop codon,splice signal, localization signal, etc.), to modify a nucleic acidsequence (e.g., introduce a mutation), and the like. As such, thecompositions can be used to modify DNA in a site-specific, i.e.,“targeted” way, for example gene knock-out, gene knock-in, gene editing,gene tagging, etc., as used in, for example, gene therapy.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, α-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

As used herein, a “regulatory gene” or “regulatory sequence” is anucleic acid sequence that encodes products (e.g., transcriptionfactors) that control the expression of other genes.

As used herein, a “protein coding sequence” or a sequence that encodes aparticular protein or polypeptide, is a nucleic acid sequence that istranscribed into mRNA (in the case of DNA) and is translated (in thecase of mRNA) into a polypeptide in vitro or in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ terminus(N-terminus) and a translation stop nonsense codon at the 3′ terminus(C-terminus). A coding sequence can include, but is not limited to, cDNAfrom eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, andsynthetic nucleic acids. A transcription termination sequence willusually be located 3′ to the coding sequence.

As used herein, a “promoter” is defined as a regulatory DNA sequencegenerally located upstream of a gene that mediates the initiation oftranscription by directing RNA polymerase to bind to DNA and initiatingRNA synthesis. A promoter can be a constitutively active promoter (i.e.,a promoter that is constitutively in an active/“ON” state), it may be aninducible promoter (i.e., a promoter whose state, active/“ON” orinactive/“OFF”, is controlled by an external stimulus, e.g., thepresence of a particular compound or protein), it may be a spatiallyrestricted promoter (i.e., transcriptional control element, enhancer,etc.)(e.g., tissue specific promoter, cell type specific promoter,etc.), and it may be a temporally restricted promoter (i.e., thepromoter is in the “ON” state or “OFF” state during specific stages ofembryonic development or during specific stages of a biological process.

As used herein, the term “gene” means the deoxyribonucleotide sequencescomprising the coding region of a structural gene. A “gene” may alsoinclude non-translated sequences located adjacent to the coding regionon both the 5′ and 3′ ends such that the gene corresponds to the lengthof the full-length mRNA. The sequences which are located 5′ of thecoding region and which are present on the mRNA are referred to as 5′non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene which are transcribed into heterogenous nuclearRNA (hnRNA); introns may contain regulatory elements such as enhancers.Introns are removed or “spliced out” from the nuclear or primarytranscript; introns therefore are absent in the messenger RNA (mRNA)transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide.

As used herein, the terms “functionally linked” and “operably linked”are used interchangeably and refer to a functional relationship betweentwo or more DNA segments, in particular gene sequences to be expressedand those sequences controlling their expression. For example, apromoter/enhancer sequence, including any combination of cis-actingtranscriptional control elements is operably linked to a coding sequenceif it stimulates or modulates the transcription of the coding sequencein an appropriate host cell or other expression system. Promoterregulatory sequences that are operably linked to the transcribed genesequence are physically contiguous to the transcribed sequence.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The term “antibody” as used herein refers to polyclonal and monoclonalantibodies and fragments thereof, and immunologic binding equivalentsthereof. The term “antibody” refers to a homogeneous molecular entity,or a mixture such as a polyclonal serum product made up of a pluralityof different molecular entities, and broadly encompassesnaturally-occurring forms of antibodies (for example, IgG, IgA, IgM,IgE) and recombinant antibodies such as single-chain antibodies,chimeric and humanized antibodies and multi-specific antibodies. Theterm “antibody” also refers to fragments and derivatives of all of theforegoing, and may further comprise any modified or derivatised variantsthereof that retains the ability to specifically bind an epitope.Antibody derivatives may comprise a protein or chemical moietyconjugated to an antibody. A monoclonal antibody is capable ofselectively binding to a target antigen or epitope. Antibodies mayinclude, but are not limited to polyclonal antibodies, monoclonalantibodies (mAbs), humanized or chimeric antibodies, camelizedantibodies, single chain antibodies (scFvs), Fab fragments, F(ab′)2fragments, disulfide-linked Fvs (sdFv) fragments, for example, asproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, intrabodies, nanobodies, synthetic antibodies, andepitope-binding fragments of any of the above.

As used herein, the term “humanized mouse” (Hu-mouse) is a mousedeveloped to carry functioning human genes, cells, tissues, and/ororgans. Humanized mice are commonly used as small animal models inbiological and medical research for human therapeutics. Immunodeficientmice are often used as recipients for human cells or tissues, becausethey can relatively easily accept heterologous cells due to lack of hostimmunity.

HSCs possess the ability of multipotency (i.e., one HSC candifferentiate into all functional blood cells) and self-renewal (i.e.,HSCs can divide and give rise to an identical daughter cell, withoutdifferentiation). Through a series of lineage commitment steps, HSCsgive rise to progeny that progressively lose self-renewal potential andsuccessively become more and more restricted in their differentiationcapacity, generating multi-potential and lineage-committed progenitorcells, and ultimately mature functional circulating blood cells.

The ability of hematopoietic stem and progenitor cells (HSPCs) toself-renew and differentiate is fundamental for the formation andmaintenance of life-long hematopoiesis and deregulation of theseprocesses may lead to severe clinical consequences. HSPCs are alsohighly valuable for their ability to reconstitute the hematopoieticsystem when transplanted and this has enabled their use in the clinic totreat a variety of disorders including bone marrow failure,myeloproliferative disorders and other acquired or genetic disordersthat affect blood cells.

As used herein, a “pluripotent cell” refers to a cell derived from anembryo produced by activation of a cell containing DNA of all female ormale origin that can be maintained in vitro for prolonged, theoreticallyindefinite period of time in an undifferentiated state that can giverise to different differentiated tissue types, i.e., ectoderm, mesoderm,and endoderm. “Embryonic stem cells” (ES cells) are pluripotent stemcells derived from the inner cell mass of a blastocyst, an early-stagepreimplantation embryo.

As used herein “pharmaceutically acceptable carrier” encompasses any ofthe standard pharmaceutical carriers, such as a phosphate bufferedsaline solution, water and emulsions such as an oil/water or water/oilemulsion, and various types of wetting agents.

This work shows that one-time hematopoietic stem and progenitor cell(HSPC) transplantation holds the potential to become a life-longcurative therapy for a disease or disorder associated with mitochondrialdysfunction. Given the risks associated with allogeneic stem celltransplantation, the objective was to develop an autologous HSPC genetherapy for mitochondrial diseases.

As discussed above, mitochondrial diseases/disorders may be caused bymutations, acquired or inherited, in mitochondrial DNA (mtDNA) or innuclear genes that code for mitochondrial components. They may also bethe result of acquired mitochondrial dysfunction due to adverse effectsof drugs, infections, or other environmental causes.

Examples of mitochondrial diseases include, but are not limited to,mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leber'shereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosingencephalopathy, Neuropathy, ataxia, retinitis pigmentosa, and ptosis(NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), MyoclonicEpilepsy with Ragged Red Fibers (MERRF), Mitochondrial myopathy,encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS),mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) anddiseases due to mitochondrial complex deficiency, such as Friedreich'sataxia (FRDA).

FRDA is a progressively lethal multi-systemic disease. Although theexact function of FXN is still under debate, it is predicted to assistin the biogenesis of mitochrondrial iron-sulfur clusters. Thus, frataxindeficiency results in altered cellular iron metabolism, increasedmitochondrial iron load, decreased mitochondrial energy production andbiogenesis as well as increased oxidative stress. Clinical featuresinclude gait and limb ataxia, muscle weakness, dysarthria and alsovision and hearing anomalies, diabetes and cardiomyopathy. Frataxindeficiency impacts neuronal functions particularly and this affectsmainly the peripheral and central nervous systems (CNS), leading to theprogressive destruction of the Dorsal Root Ganglia (DRG). Thisprogressive neurodegeneration leads to loss of motor skills andprogressive muscle degeneration, and ultimately inability to walk within10 to 15 years of onset. Heart abnormalities cause premature death in60% to 80% of the affected individuals; the average age of death is inthe mid-thirties. The different clinical trials of pharmacologicalcompounds against oxidative stress (idebone and Coenzyme Q 10) ormitochondrial iron accumulation (deferipone) failed to prove efficacy.An epigenetic approach using an histone deacetylase inhibitor iscurrently being testing in phase I clinical trial.

Hematopoietic stem and progenitor cells (HSPCs) are ideal candidates foruse in regenerative medicine and cell replacement therapies because oftheir ease of isolation, self-renewal capacity, and safety. As such, thepresent disclosure evaluates the impact of hematopoietic stem andprogenitor cell (HSPC) transplantation in a mouse model of FRDA. Therationale for using HSPC to treat FRDA came from previous work oncystinosis, a multi-systemic lysosomal storage disorder. Briefly, HSPCtransplantation using a self-inactivating (SIN)-lentivirus vectorcontaining human CTNS cDNA under the control of the strong ubiquitousshort intron-less human Elongation Factor 1 alpha (EFS) promoter inlethally irradiated Ctns^(−/−) mice (mouse model of cystinosis) led tothe abundant engraftment of HSPC-derived cells in all organs, whichcorrelated with the dramatic reduction in tissue cystine levels (up to94% decrease). This treatment also led to long-term preservation of thekidney structure and function, rescue of the eye defects and thyroiddysfunction. These data showed that a single HSPC transplant couldprevent the multi-organ failure for the lifespan of the mice. However,these results were particularly surprising as cystinosin is aubiquitous, lysosomal transmembrane protein. Addressing the cellularmechanism, it was demonstrated that transplanted HSPCs led to thetransfer of cystinosin-bearing lysosomes via tunneling nanotubes (TNTs)after differentiating into macrophages. In vivo, macrophage-derivedtubular extensions penetrated the dense tubular basement membrane anddelivered cystinosin-containing lysosomes into the epithelia inCtns^(−/−) mice, so as to prevent proximal tubule degeneration. The samemechanism has been demonstrated in the eye and thyroid ofHSPC-transplanted Ctns^(−/−) mice.

However, in contrast to the CTNS gene, overexpression of frataxin istoxic. Thus, one strategy is to generate a new lentiviral construct inwhich FXN will be expressed under the control of its own promoter andtest the efficacy and safety of this strategy in vitro and in vivo.Alternatively, or in addition thereto, removing the trinucleotideextension mutation using gene editing techniques is contemplated tocorrect the defect in FRDA HSPC.

Accordingly, in one aspect, the invention provides a method of treatinga mitochondrial disease or disorder in a subject. The method includesintroducing ex vivo a functional human frataxin (hFXN) intohematopoietic stem and progenitor cells (HSPCs) of the subject, andthereafter transplanting the HSPCs into the subject, thereby treatingthe mitochondrial disease or disorder. The step of introducing mayinclude contacting a vector comprising a polynucleotide encoding hFXNand an ubiquitous or endogenous FXN promoter with the HSPCs and allowingexpression of hFXN. In various embodiments, the vector is aself-inactivating (SIN)-lentivirus vector, such as pCCL-EFS-FXN orpCCL-FRDAp-FXN. In various embodiments, expression of hFXN correctsneurologic, cardiac and muscular complications within about 6-12 monthspost-transplantation.

Nucleic acid sequences for human and mouse frataxin (FRDA) are known inthe art. See, for example, GenBank Accession No.: U43747.1, humanfrataxin mRNA, complete cds, which provides the nucleic acid sequence(SEQ ID NO: 1):

TTTACAGGGCATAACTCATTTTATCCTTACCACAATCCTATGAAGTAGGAACTTTTATAAAACGCATTTTATATNCAAGGGCACAGAGAGGNTAATTAACTTGCCCTCTGGTCACACAGCTAGGAAGTGGGCAGAGTACAGATTTACACTAGGCATCCGTCTCCTGNCCCCACATANCCAGCTGCTGTAAACCCATACCGGCGGCCAAGCAGCCTCAATTTGTGCATGCACCCACTTCCCAGCAAGACAGCAGCTCCCAAGTTCCTCCTGTTTAGAATTTTAGAAGCGGCGGGCCACCAGGCTGCAGTCTCCCTTGGGTCAGGGGTCCTGGTTGCACTCCGTGCTTTGCACAAAGCAGGCTCTCCATTTTTGTTAAATGCACGAATAGTGCTAAGCTGGGAAGTTCTTCCTGAGGTCTAACCTCTAGCTGCTCCCCCACAGAAGAGTGCCTGCGGCCAGTGGCCACCAGGGGTCGCCGCAGCACCCAGCGCTGGAGGGCGGAGCGGGCGGCAGACCCGGAGCAGCATGTGACTCTCGGGCGCCGCGCAGTAGCCGGCCTCCTGGCGTCACCCAGCCCGGCCCAGGCCCAGACCCTCACCCGGGTCCCGCGGCCGGCAGAGTTGGCCCCACTCTGCGGCCGCCGTGGCCTGCGCACCGACATCGATGCGACCTGCACGCCCCGCCGCGCAAGTTCGAACCAACGTGGCCTCAACCAGATTTGGAATGTCAAAAAGCAGAGTGTCTATTTGATGAATTTGAGGAAATCTGGAACTTTGGGCCACCCAGGCTCTCTAGATGAGACCACCTATGAAAGACTAGCAGAGGAAACGCTGGACTCTTTAGCAGAGTTTTTTGAAGACCTTGCAGACAAGCCATACACGTTTGAGGACTATGATGTCTCCTTTGGGAGTGGTGTCTTAACTGTCAAACTGGGTGGAGATCTAGGAACCTATGTGATCAACAAGCAGACGCCAAACAAGCAAATCTGGCTATCTTCTCCATCCAGTGGACCTAAGCGTTATGACTGGACTGGGAAAAACTGGGTGTTCTCCCACGACGGCGTGTCCCTCCATGAGCTGCTGGCCGCAGAGCTCACTAAAGCCTTAAAAACCAAACTGGACTTGTCTTGGTTGGCCTATTCCGGAAAAGATGCTTGATGCCCAGCCCCGTTTTAAGGACATTAAAAGCTATCAGGCCAAGACCCCAGCTTCATTATGCAGCTGAGGTGTGTTTTTTGTTGTTGTTGTTGTTTATTTTTTTTATTCCTGCTTTTGAGGACACTTGGGCTATGTGTCACAGCTCTGTACAAACAATGTGTTGCCTCCTACCTTGCCCCCAAGTTCTGATTTTTAATTTCTATGGAAGATTTTTTGGATTGTCGGATTTCCTCCCTCACATGATACCCCTTATCTTTTATAATGTCTTATGCCTATACCTGAATATAACAACCTTTAAAAAAGCAAAATAATAAGAAGGAAAAATTCCAGGAGGGGenBank Accession No.: U43747.1:526-1158, human frataxin mRNA, completecds, which provides the nucleic acid sequence (SEQ ID NO: 2):

ATGTGGACTCTCGGGCGCCGCGCAGTAGCCGGCCTCCTGGCGTCACCCAGCCCGGCCCAGGCCCAGACCCTCACCCGGGTCCCGCGGCCGGCAGAGTTGGCCCCACTCTGCGGCCGCCGTGGCCTGCGCACCGACATCGATGCGACCTGCACGCCCCGCCGCGCAAGTTCGAACCAACGTGGCCTCAACCAGATTTGGAATGTCAAAAAGCAGAGTGTCTATTTGATGAATTTGAGGAAATCTGGAACTTTGGGCCACCCAGGCTCTCTAGATGAGACCACCTATGAAAGACTAGCAGAGGAAACGCTGGACTCTTTAGCAGAGTTTTTTGAAGACCTTGCAGACAAGCCATACACGTTTGAGGACTATGATGTCTCCTTTGGGAGTGGTGTCTTAACTGTCAAACTGGGTGGAGATCTAGGAACCTATGTGATCAACAAGCAGACGCCAAACAAGCAAATCTGGCTATCTTCTCCATCCAGTGGACCTAAGCGTTATGACTGGACTGGGAAAAACTGGGTGTTCTCCCACGACGGCGTGTCCCTCCATGAGCTGCTGGCCGCAGAGCTCACTAAAGCCTTAAAAACCAAACTGGACTTGTCTTGGTTGGCCTATTCCGGAAAAGATGCTTGA,GenBank Accession No.: U95736.1, Mus musculus frataxin mRNA, completecds, which provides the nucleic acid sequence (SEQ ID NO: 3):

CGGCCGCGGAGCTGGAGTAGCATGTGGGCGTTCGGAGGTCGCGCAGCCGTGGGCTTGCTGCCCCGGACGGCGTCCCGGGCCTCCGCCTGGGTCGGGAACCCGCGCTGGAGGGAACCGATCGTAACCTGCGGCCGCCGAGGCCTACATGTCACAGTCAACGCCGGCGCCACCCGCCACGCCCATTTGAACCTCCACTACCTCCAGATTCTGAACATCAAAAAGCAGAGCGTCTGCGTGGTGCATTTGAGGAACTTGGGGACATTGGACAACCCAAGCTCTCTAGACGAGACAGCGTATGAAAGACTGGCGGAAGAGACCCTGGACTCCCTGGCCGAGTTCTTTGAAGACCTCGCAGACAAGCCCTATACCCTGGAGGACTACGATGTCTCTTTTGGGGATGGCGTGCTCACCATTAAGCTGGGCGGGGATCTAGGGACCTACGTGATCAACAAGCAGACCCCAAACAAGCAAATCTGGCTGTCTTCTCCTTCCAGCGGCCCCAAGCGCTATGACTGGACCGGGAAGAACTGGGTGTACTCTCATGACGGCGTGTCTCTGCATGAGCTGCTGGCCAGGGAGCTGACTAAAGCTTTAAACACCAAACTGGACTTGTCTTCATTGGCCTATTCTGGAAAAGGCACTTGACTGCCAGCCAGATTCCAAGACATTAAACACTGTCAGGTGAAGACCCCCAGCCTCCTCCTGTAGCTGAATGTCTGCCTTCCCATACCTGCTCCTGAAGATAGTCACACCGTGTGTGACAGCTCTGTGAAAAAAGTGTGTTCCCTCCCACCCTGTCCCCGGACCTGGCTCTTCATTTCTACAGACATTTGTTAGGATTATGTCATTTGCTCCCCAACCTGAGACCTCTGGTCTCTTAGAAAGTCTTATATGCTGGGCAGTGGTGGCGCACGCCTTTAATCCCAGCACTCGGGAGGCAGAGGCAGGCGGATTTCTGAGTTGGAGGCCAGCCTGGTTTACAGAGTGAGTTCCAGGACAGCCAGGACTACACAGAGAAACCCTGTGTCGAAAAAAAAAAAAAAAAAAAGAAAGAAAGAAAGTCTTACACCACAAGTGTGTCCATGATATAACAGCC,and GenBank Accession No.: U95736.1:22-645 Mus musculus frataxin mRNA,complete cds, which provides the nucleic acid sequence (SEQ ID NO: 4):

ATGTGGGCGTTCGGAGGTCGCGCAGCCGTGGGCTTGCTGCCCCGGACGGCGTCCCGGGCCTCCGCCTGGGTCGGGAACCCGCGCTGGAGGGAACCGATCGTAACCTGCGGCCGCCGAGGCCTACATGTCACAGTCAACGCCGGCGCCACCCGCCACGCCCATTTGAACCTCCACTACCTCCAGATTCTGAACATCAAAAAGCAGAGCGTCTGCGTGGTGCATTTGAGGAACTTGGGGACATTGGACAACCCAAGCTCTCTAGACGAGACAGCGTATGAAAGACTGGCGGAAGAGACCCTGGACTCCCTGGCCGAGTTCTTTGAAGACCTCGCAGACAAGCCCTATACCCTGGAGGACTACGATGTCTCTTTTGGGGATGGCGTGCTCACCATTAAGCTGGGCGGGGATCTAGGGACCTACGTGATCAACAAGCAGACCCCAAACAAGCAAATCTGGCTGTCTTCTCCTTCCAGCGGCCCCAAGCGCTATGACTGGACCGGGAAGAACTGGGTGTACTCTCATGACGGCGTGTCTCTGCATGAGCTGCTGGCCAGGGAGCTGACTAAAGCTTTAAACACCAAACTGGACTTGTCTTCATTGGCCTATTCTGGAAAAGGCACTTGA.

In another aspect, the method of treating a mitochondrial disease ordisorder in a subject includes contacting cells expressing hFXN from thesubject with a vector encoding a gene editing system that whentransfected into the cells removes a trinucleotide extension mutation ofendogenous hFXN, thereby treating the mitochondrial disease or disorder.In various embodiments, the gene editing system is selected from thegroup consisting of CRISPR/Cas, zinc finger nucleases, and transcriptionactivator-life effector nucleases. The step of contacting may beperformed ex vivo by first obtaining a sample of cells from the subject,transfecting the gene editing system into the sample of cells, andthereafter transplanting the transfected cells into the subject, therebytreating the mitochondrial disease or disorder. The sample of cells maybe any cells expressing hFXN, such as, for example, blood cells or HSPCsof the subject.

In addition to lysosomes, mitochondria can readily be transferred viatunneling nanotubes (TNTs). Using the YG8R mouse model, it was thereforetested if HSPC transplantation could rescue FRDA. The premise is thatmitochondrial cross-correction would occur in all injured tissues viaTNTs generated by HSPC-derived macrophages. YG8R mice are currentlyconsidered the best animal model of FRDA as they express only the humanmutated frataxin containing 280 GAA repeats (SEQ ID NO: 14), withoutendogenous murine frataxin, fxn^(−/−) FXN⁺. This mouse model exhibits adecrease of 57% frataxin expression resulting in a mild progressivephenotype including ataxia, and coordination and locomotor anomaliessimilar to the clinical manifestations in FRDA patients. The micedisplay a degeneration of the large sensory neurons of DRG, and decreasein aconitase activity and increase of oxidized proteins in the brain,heart and skeletal muscle. Thus, the advantages of this mouse model,compared to tissue-specific conditional FXN knockout models for FRDA,are that the genetic defect is similar to that of humans and that theimpact of stem cell therapy is tested in the CNS, heart and skeletalmuscle in the same animal model. The impact of HSPC transplantation inYG8R mice has been impressive as the neurological complications andmuscle weakness were fully rescued in the treated mice, with functional,histological and biochemical properties comparable to wild-type (WT)mice.

The present disclosure also demonstrates that HSPCs differentiated intophagocytic cells in the brain, spinal cord, DRG, muscle and heart andtransferred frataxin to the adjacent disease cells. These data representthe first proof of concept that FRDA can be treated by HSPCtransplantation and the first treatment strategy resulting inphysiologic rescue of the complications associated with FRDA in a mousemodel.

Given the high risk of morbidity and mortality associated withallogeneic HSPC transplantation, it remains an uncertain therapeuticchoice for many diseases after consideration of the risk/benefit ratio.The major complication is graft-versus-host disease (GVHD), acute GVHDgrade II-IV occurred in 20% to 32% of patients and chronic GVHD in 16%to 59%, both significantly impacting survival of the recipients.Moreover, high risks of infection related to the myeloablative regimenand immunosuppressive medications account for 16% to 19% of deaths.Since it avoids the risks of immune rejection and GVHD, autologous HSPCtransplantation is a safer approach. Thus, in the case of cystinosis, anautologous HSPC transplantation was developed using a self-inactivated(SIN)-lentivirus vector (LV) containing human CTNS cDNA and tested thisstrategy in the Ctns^(−/−) mice. It was therefore shown that transducedcells were capable of decreasing cystine content in all tissues and ledto kidney function improvement. In vitro studies using human CD34+ HSPCsisolated from peripheral blood of healthy donors and cystinosis patientshave now completed, and the serial transplantation in the Ctns^(−/−)mice has been significantly advanced.

Accordingly, the present disclosure provides a method for autologoustransplantation of ex vivo gene-modified HSPCs to introduce a functionalfrataxin. In various embodiments, the method involves use of a pCCLSIN-LV vector or gene editing to remove a trinucleotide extensionmutation of endogenous hFXN in the HSPCs. As demonstrated herein, thisapproach has proven effective in the YG8R mouse model. This represents aunique treatment approach for FRDA that should lead to a clinical trialfor this disease after completing the pharmacology/toxicology studies.Gene therapy approaches for FRDA have already been tested in vitro andin vivo with successful outcomes. Infection of human fibroblasts derivedfrom FRDA patients with different viral vectors, adeno-associated virus(AAV), LV or herpes simplex virus type 1 (HSV-1), containing human FXN(hFXN) cDNA or full genomic DNA resulted in the partial or completerestoration of the WT cellular phenotype in response to oxidativestress. Human FXN cDNA delivery in the nervous system of conditionalneuronal fxn-knockout mice using HSV-1 vector led to the completerecovery in motor coordination. Intraperitoneal injection of AAV-9vector containing hFXN cDNA in the cardiac and skeletal muscleconditional frataxin-knockout mouse model (MCK mice), doubled the lifespan of the mice and improved their cardiac function. It has beenrecently reported that complete prevention and reversal of severecardiomyopathy in MCK mice by has been achieved by intravenous injectionof AAV9-hFXN cDNA.

In contrast to the gene therapy approaches tested so far for FRDA, thepresent disclosure provides use of a SIN-LV or gene editing to correctHSPCs for a systemic therapeutic strategy. Vectors derived fromlentiviruses have supplanted γ-retroviral vector for gene therapy due totheir superior gene transfer efficiency and better biosafety profile.Indeed, all cases of leukemogenic complications observed to date inclinical trials or animal models involved the use of retroviral vectorswith LTR containing strong enhancer/promoters that can trigger distantenhancer activation. In contrast, the third-generation of lentivirusvectors, SIN-LV, with the deletions in their LTR, contains only oneinternal enhancer/promoter, which reduces the incidence of interactionswith nearby cellular genes, and thus, decreases the risk of oncogenicintegration. SIN-LV are also designed to prevent the possibility ofdeveloping replication competent lentivirus (RCL) during production ofviral supernatants with three packaging plasmids necessary forproduction. Lentivirus vectors efficiently transduce HSPCs and do notalter their repopulation properties, which make this type of vector anattractive vehicle for stem cell gene therapy.

Clinical trials using SIN-LV to gene-correct human HSPCs are beingundertaken in the U.S. and Europe for several conditions includingHIV-1, β-thalassemia, immune deficiencies, metabolic diseases andcancers. For immune deficiency disorders, 35 patients have beentransplanted with SIN-LV-modified HSPCs so far. A clinical trial inpatients with Adrenoleukodystrophy (ALD) has achieved stable genecorrection in ˜20% of hematopoietic cells in two patients. Cerebraldemyelination was arrested without further progression over three yearsof follow-up, which represents a clinical outcome comparable to thatobserved after allogeneic transplantation; there was no evidence ofclonal dominance. Recently, a clinical trial for Wilskott-Aldrichsyndrome was reported in three patients 32 months post-transplantation.Stable and long-term engraftment of the gene-modified HSPCs (25-50%)resulted in improved platelet counts, protection from bleeding andinfections, and resolution of eczema. Another clinical success wasrecently reported in three pre-symptomatic patients with MetachromaticLeukodystrophy. Transduced cell-derived blood cell engraftment achieved45 to 80%, and up to 24 months later, protein activity was reconstitutedto above normal values in cerebrospinal fluid associated with a cleartherapeutic benefit.

Because Friedreich's ataxia is a monogenic disease caused by a shortageof the frataxin protein, gene therapy appears to be a promisingalternative treatment. The recent gene therapy successes using AAVvectors in the MCK mice not only prevented heart failure when given topresymptomatic animals, but also reversed the cardiomyopathy when givenafter the onset. While encouraging, this approach presents potentialsafety and logistic concerns: i) localized delivery by direct viralinjection to affected sites poses certain challenges in accessing sitessuch as heart and brain and leads only to tissue-specific rescue, ii)systemic AAV delivery remains difficult in humans due to the high levelsof vector necessary, leading to vector synthesis and safety concerns. Incontrast, HSPC gene therapy approach has the key advantages: i) ittreats all the complications by a single infusion of stem cells, ii)gene-correction will occur ex vivo in a controlled environment allowingcell characterization prior to transplantation, iii) gene-correctedHSPCs will reside in the bone marrow niche after transplantation wherethey will self-renew and become a reservoir of healthy cells for thelifespan of the patients, iv) it avoids immune reaction as compared toallogeneic transplantation. Thus, autologous HSPC gene therapy couldprovide a cure for the lethal disease FRDA for which no treatmentcurrently exists.

Another innovative aspect provided herein is the use of HSPCs asdelivery vehicles for functional mitochondrial genes. Many diseases suchas metabolic, cancer, cardiovascular and neurodegenerative disorders areassociated with mitochondrial dysfunction. Inherited mitochondrialdiseases are relatively frequent and affect 1 in every 5,000 children,often causing fatal illnesses. While many attempts have been made todeliver healthy mitochondria to diseased cells and tissues, the efficacyof such approaches has been limited and usually short-term.

The present disclosure demonstrates that one single systemictransplantation of WT HSPCs in young adult YG8R mice fully prevents thedevelopment of FRDA pathology including neurobehavioral deficits, muscleweakness and degeneration of DRG sensory neurons. One advantage ofexogenous HSPC transplantation is the capacity of these cells topermanently replace/repopulate the marrow and migrate from their nicheto differentiate into phagocytic cell types within multiple diseasedtissues. HSPCs can even transmigrate across the blood brain barrier andengraft within the CNS as differentiated microglia. This phenomena isenhanced by tissue injury and even by the use of busulfan-mediatedmyeloablation, as opposed to total body irradiation, which enhances theclinical relevance of this work for the treatment of FRDA. Consistently,it has been shown that transplanted HSPCs differentiate into microglialcells within the CNS of the YG8R mice but also macrophages in DRG,peripheral nerves, skeletal muscle and heart, the primary sites of FRDApathological complications.

Restoration of mitochondrial function in WT HSPC-treated mice ascompared to YG8R controls was evidenced by biochemical, molecular andhistological studies. First, significant reduction in oxidative stresswas observed in WT HSPC-treated YG8R tissues as compared to controllittermates. Oxidative stress is a major component in FRDA pathogenesisand likely to account for neuronal preservation. Oxidative stress hasalso recently been shown to induce DNA damage and elevation of Poly(ADP-Ribose) Polymerase-1 (PARP-1) expression in frataxin-deficientmicroglial cells, which increased microglial activation. Because PARP1activation leads to increased inflammatory cytokine expression inmicroglial cells, these findings suggest that oxidative stress mayinduce neuroinflammatory-mediated neurodegeneration in FRDA. Hence, therobust neurological phenotype rescue demonstrated herein in HSPC-treatedYG8R may partially be due to the replacement of the frataxin-deficientmicroglial cells by wild-type microglia, another potential advantage ofthis therapeutic strategy. Mitochondrial function was also assessed bymitochondrial PCR array profiling in the cerebrum of the mice. Thefindings provided herein show largely upregulated genes >2 fold changein YG8R mice compared to WT (13 genes out of 84 total) while very fewchanges were identified between WT and YG8R/WT HSPCs mice (4 genes) andfor none the difference was significant. The significantly upregulatedgenes in YG8R vs WT include three solute mitochondrial carrier family 25genes, Mipep, an important component of the human mitochondrial importmachinery implicated in developmental delay and the fatty acidtransporter Cpt1b, which is upregulated in stress and Post-TraumaticStress Disorder. Finally, cellular iron metabolism dysregulation isevidenced in FRDA by the presence of iron deposits in cardiomyocystes ofpatients (Lamarche, et al. Lemieux, The cardiomyopathy of Friedreich'sataxia morphological observations in 3 cases. The Canadian journal ofneurological sciences. Le journal canadien des sciences neurologiques 7,389-396 (1980)). Similarly, the present disclosure demonstrates thepresence of abundant iron deposition in heart sections from YG8Rcontrols while very few were observed in WT and YG8R/WT HSPCs mice,suggesting normal iron metabolism in the treated YG8R mice. In contrast,preclinical and clinical data using an iron chelator are sometimesopposite in function of the dosage (Pandolfo, et al., Deferiprone forthe treatment of Friedreich's ataxia. J Neurochem 126 Suppl 1, 142-146(2013)). These data demonstrate correction of mitochondrial function inthe different affected tissues in FRDA, brain, skeletal muscle andheart, after one single systemic transplantation of WT HSPCs.

The data provided herein strongly suggest that frataxin cross-correctionmechanism is involved in FRDA phenotype rescue after WT HSPCtransplantation. Indeed, the evidence demonstrates abundant transfer ofthe mitochondrial frataxin from the HSPC-derived microglia/macrophagesto neurons in brain, spinal cord, and DRGs, and myocytes in skeletalmuscle and heart. The data also demonstrates the transfer of thenon-related mitochondrial protein Cox8, showing non-selective transferof mitochondrial proteins occur.

As discussed above, it has previously been reported that HSPC-derivedmacrophages engrafted in kidney could deliver cystinosin-containinglysosomes to proximal tubular cells via TNTs in the mouse model ofcystinosis. In this context, TNTs crossing the basement membrane was theonly route possible across the continuous, thick, dense tubular basementmembrane to access the tubular cells. Transfer of mitochondria via TNTshas previously been shown in vitro in response to cellular stress, andthis prompted the testing of HSPC transplantation in FRDA. Here, it hasbeen shown in culture that frataxin-bearing mitochondria could betransferred via TNT intercellular connections from macrophages tofrataxin-deficient cells. In vivo, it has been observed that themitochondrial proteins frataxin and Cox8 conjugated with GFP within hostneurons, demonstrating neuronal cross-correction from microglial cells,which is efficient as about 50% of neurons contained Cox8-GFP in thespinal cord. Several routes have to be considered for this transfer: i)Vesicular exchange of genetic material, messenger RNAs were shown to betransferred from graft-derived microglia to neurons via extracellularvesicles/exosome shedding; ii) Release of mitochondria-containingvesicles, this was previously shown from mesenchymal stem cells topulmonary alveoli in acute lung injury model, or more recently fromastrocytes to neurons in a cerebral ischemia model; iii)Microglia-to-neuron transfer of mitochondria via the microglial branchextensions directly in contact with neurons. While this route has notyet been considered, the data presented herein suggest that this is apossible mode of transfer. Indeed, it has been shown that themitochondrial proteins Cox8-GFP and FXN-GFP were transferred to neuronsand that GFP punctae were also present within the DsRed⁺ microglialbranch extensions. Moreover, it has been shown that most of the neuronscontaining GFP⁺ mitochondria were in contact with the DsRed⁺ microglialbranch extensions. Microglial processes are dynamic, actively retractingand expanding, and capable of making direct contact with neurons,especially in context of injury, during which the duration of thecontact is prolonged, supporting this hypothesis.

Thus, this strategy turns HSPCs into intelligent and widespread deliveryvehicles to obtain stable and sustained cross-correction after theirdifferentiation into microglia/macrophages in the brain, spinal cord,DRG, skeletal muscle and heart. This work also demonstrates the transferof frataxin from LV-hFXN-GFP-transduced HSPCs to diseased neurons andrepresents the first proof of concept for the development of a HSPC genetherapy strategy for mitochondrial disorders such as FRDA.

The following examples are intended to illustrate but not limit theinvention.

Example 1 Treatment of FRDA Mouse Model Using HPSC Transplantation

Systemic transplantation of wild-type HSPCs prevents onset of locomotordeficits in YG8R mice. To assess the effects of HSPC transplantation onFRDA, the YG8R mouse model expressing the mutant human FXN genecontaining 280 GAA repeats (SEQ ID NO: 14), and lacking endogenousmurine frataxin, mfxn^(−/−) hFXN⁺ was used. Lethally irradiated 2month-old YG8R mice were transplanted with wild-type (WT) GFP-expressingHSPCs (n=13) and donor-derived blood cell engraftment ranged from 35 to96% as determined by flow cytometry. Mice are sacrificed for analysis at7 months post-transplantation, i.e., at 9 months of age. As controls, WTlittermates (n=17), untreated YG8R (n=4) or lethally irradiated YG8Rmice transplanted with mfxn^(−/−) hFXN⁺ HSPCs (n=5) were analyzed. Allthe mice were assessed by behavioral testing at 5 months old (3 monthspost-transplant), and 8 WT, 4 YG8R (3 untreated and 1 transplanted withmfxn^(−/−) hFXN⁺ HSPCs) and 3 YG8R mice transplanted with WT HSPCs wereanalyzed at 9 months old.

Progressive neurodegeneration in FRDA patients leads to loss of motorskills and progressive muscle degeneration. The YG8R mouse replicateshuman FRDA neurological symptoms such as coordination deficits fromthree months of age with a progressive decrease in locomotor activity.Thus, the effect of HSPC transplantation on performance of motor- andsensory-dependent functional tasks and on muscle strength at both 5 and9 months of age was assessed (3 and 7 months post-transplantation,respectively). No difference was observed in performance in any of thebehavioral tests at either time point between untreated YG8R mice andthose transplanted with mfxn^(−/−) hFXN⁺ HSPCs, indicating that neitherirradiation nor transplantation with mfxn^(−/−) hFXN⁺ HSPCs amelioratethe disease phenotype. Compared to WT mice, YG8R mice (controls) andYG8R mice transplanted with mfxn^(−/−) hFXN⁺ HSPCs displayedsignificantly reduced open field locomotor activity, impairedcoordination on rotarod, and alterations in gait as well assignificantly decreased forelimb grip strength at both time points (FIG.1A). In contrast, YG8R mice transplanted with WT HSPCs exhibited normallocomotor activity and muscle strength at both 3 and 7 monthspost-transplantation (FIG. 1A). Interestingly, and in contrast toprevious findings in the cystinosis model, the YG8R mouse exhibiting thelowest level of donor-derived blood cell engraftment still exhibitedphysiological rescue of the neurobehavioral deficits. Together, thesedata demonstrate that HSPC transplantation in 2-month-old YG8R micecompletely rescued the progressive neurobehavioral and muscular deficitscharacteristic of this FRDA animal model.

Neurodegeneration in FRDA involves primarily the sensory components ofthe central nervous system (CNS) and peripheral nervous system (PNS),beginning with loss of large sensory neurons in the dorsal root ganglia(DRG). Loss of sensory neurons in DRGs also occurs in YG8R mice and ischaracterized by the presence of large vacuoles. In 9-month-old controlYG8R mice, vacuolar accumulation in L5 DRG neurons was detected with nosignificant difference in vacuole area between non-treated andmfxn^(−/−) hFXN⁺ HSPC-transplanted YG8R mice (FIG. 1C). In contrast,YG8R mice treated with WT HSPCs exhibited a significantly reducedvacuolar area that was comparable to WT mice (FIG. 1C). These datademonstrate that early transplantation of HSPCs prevents thedegeneration of sensory neural cell bodies of the DRG in YG8R mice.

HSPCs Differentiate into Phagocytic Cells after Engraftment in theNervous System.

Because FRDA affects the central nervous system (CNS) in addition toperipheral sensory neurons, the engraftment and differentiation of HSPCswas investigated in different regions of the nervous system. It wasfound that substantial engraftment of GFP⁺ HSPC-derived cells within theDRGs, spinal cord and peripheral nerves (FIGS. 1C and 5). Within DRGs atall levels, donor cells were found in close proximity to neurons andwere immunoreactive for the macrophage markers CD68 and MHCII, as wellas Iba1, characterizing these cells as DRG resident macrophages (FIGS.1D, 1E, 6A and 6B). In the spinal cord, HSPC-derived cells were abundantin the ascending sensory axon tracts, within the dorsal and ventralroots, motor pools and dorsal spinal cord gray matter (FIGS. 1C and 1D).These cells were >99% Iba1⁺ and CD68⁺, while fewer cells expressed MHCII(˜30%; FIGS. 6A-6C) indicating their microglial identity.3D-visualization of engrafted spinal cord subjected to tissue clearingshowed that a high concentration of engrafted HSPC-derived cells wasfound in close proximity to perivascular regions, suggesting that thesecells infiltrate the CNS via the vasculature.

Graft-derived cells were also detected throughout gray and white matterin the brain, brainstem and cerebellum in treated YG8R mice (FIG. 2A).The vast majority (>99%) of HSPC-derived cells within all regions of thebrain displayed the typical ramified morphology of microglia andexpressed CD68 and Iba1, but were not immunoreactive for MHCII,demonstrating that these cells were microglial cells (FIGS. 2B, 6A, 6Band 6D). Perivascular infiltration in the brain was further demonstratedby the presence of GFP⁺ HSPC-derived cells in close proximity of bloodvessels (FIG. 6E) especially in the highly vascularized choroid plexus(FIG. 6F).

WT HSPC transplantation restores frataxin expression and mitochondrialfunction in the brain of YG8R mice. Murine frataxin (mFxn) expressionanalysis in the brain confirmed that tissue engraftment of theHSPC-derived cells correlated with partial restoration of mfxnexpression in treated mice as compared to YG8R controls, although not upto WT expression levels; a residual expression was also detected in YG8Rmice likely due to cross-reactivity with human FXN (FIG. 2C).Mitochondrial dysfunction in FRDA is associated with the presence ofincreased levels of oxidized proteins within tissues. Compared to WTcontrols, levels of oxidized proteins were significantly higher in thecerebrum of YG8R mice and YG8R mice transplanted with mfxn^(−/−) hFXN⁺HSPCs (FIG. 2D). WT HSPC transplantation resulted in significantattenuation of oxidized protein levels in YG8R mice to a levelcomparable to WT, suggesting restoration of mitochondrial function intreated mice (FIG. 2D).

Additionally, mitochondrial function was assessed using mitochondrialPCR array profiling in the cerebrum of WT, YG8R, and YG8R/WT HSPCs.Expression of numerous mitochondrial genes crucial to a wide variety ofprocesses ranging from control of apoptosis to oxidative phosphorylationwere altered in the YG8R animals; out of 89 genes tested, 15.7% had atan increase of at least two-fold over WT, while only 4.4% wereupregulated in treated animals (FIG. 2E). Of these genes, five weresignificantly upregulated genes were found in YG8R mice compared to WT,including several members of the SLC family of inner mitochondrialmembrane transporters as well as other proteins involved inmitochondrial lipid metabolism (FIG. 2E). No significant difference wasevidenced between YG8R/WT HSPCs and WT mice (FIG. 2E). The PCR arraydata findings reflect significant mitochondrial dysfunction in YG8R micethat is corrected in the WT HSPC-treated YG8R mice.

HSPCs Engraft Abundantly in Heart and Muscle of YG8R Mice, RestoreMitochondrial Function and Improve Skeletal Muscle Atrophy.

Increased oxidized proteins was also demonstrated in skeletal muscle ofYG8R controls (YG8R and YG8R/YG8R HSPCs; p=0.0798) relative to WT mice,although not significant, and normal level was found in the treated YG8Rmice (FIG. 3A). Furthermore, lactate and pyruvate levels were measuredby mass spectrometry analysis of skeletal muscle biopsies, a commonassay for measuring impairment in oxidative metabolism, which was shownto be elevated in some mitochondrial diseases. A significant increase oflactate and lactate-to-pyruvate ratio in skeletal muscle of YG8R micewas demonstrated compared to WT mice, which was corrected in thetransplanted WT HSPC-transplanted YG8R mice (FIG. 3B). These datarepresent further evidence of mitochondrial dysfunction in the YG8Rmice, which is normalized in the treated mice.

In addition to neurological deficits, FRDA patients also develop aprogressive hypertrophic cardiomyopathy. Thus, the potential impact ofHSPC transplantation on heart pathology in YG8R mice was investigated.However, as cardiomyopathy is very mild in this mouse model, nosignificant phenotype was found in the YG8R mice compared to WT at 9months of age. A significant indicator of cellular iron metabolismdysregulation is the presence of iron deposits. Iron deposits incardiomyocytes were observed in FRDA patients and in old (14-18 months)YG22 mice. Perl's staining of heart sections did not reveal any irondeposit in 9-month old YG8R mice as expected. Thus, the test wasperformed in older mice (18 month old), and iron deposition incardiomyocytes were present in the non-treated YG8R or transplanted withYG8R HSPCs mice, while significantly decreased in YG8R/WT HSPCs mice(FIG. 3C). These data show the capacity of WT HSPC transplantation tocorrect mitochondrial iron metabolism in YG8R mice.

In both heart and skeletal muscle tissues, levels of mFxn expressionwere increased in the WT HSPC-treated mice compared to YG8R controls(FIGS. 3D and 3E) and confocal microscopy analysis revealed a high levelof GFP⁺ cells engrafted in these tissues in HSPC-transplanted YG8Ranimals (FIGS. 3F and 3G). The engrafted GFP⁺ cells expressed CD68 andMHCII (FIGS. 7A and 7B), indicating that these cells are macrophages.Taken together, these data indicate that HSPC-derived cells integrateinto the heart and skeletal muscle and differentiate into macrophages inYG8R mice.

Muscle strength was also observed to be significantly impaired in YG8Rmice and normal in the WT HSPC-transplanted YG8R mice. To investigatepotential muscular atrophy in YG8R mice, the expression levels weremeasured of two muscle-specific E3 ibiquitin lagases, Muscle RING finger1 (MuRF-1) and F-box (MAFbx)/atrogin-1, and a member of the transforminggrowth factor-β superfamily, myostatin, which are increased in each typeof skeletal muscle atrophy. MuRF-1, atrogin-1 and myostatin expressionwas increased in skeletal muscle from YG8R mice compared to WT (althoughnot significant for Atrogin 1), whereas the levels were normal in thetreated YG8R mice (FIG. 3H), demonstrating the rescue of this defect byHSPC transplantation.

Macrophages deliver frataxin-bearing mitochondria to diseased cells viatunneling nanotubes in vitro. It has been previously reported in thecontext of the lysosomal storage disorder cystinosis, that HSPC-derivedmacrophages promote functional rescue of diseased cells through alysosomal cross-corrective mechanism via TNTs. Hence, it wasinvestigated whether phagocytic cells could also mediate the transfer offrataxin-bearing mitochondria into mfxn^(−/−) hFXN⁺ cells via similarroute. Fibroblasts harvested from YG8R neonate skin were co-culturedwith macrophages isolated from the bone marrow of Cox8-GFP DsRed mice,ubiquitously expressing the mitochondrial Cox8 protein fused to GFPalongside the cytosolic DsRed reporter gene. Using live imaging, it wasobserved that GFP⁺ mitochondria were transferred from theDsRed-expressing macrophages to the mfxn^(−/−) hFXN⁺ fibroblasts vialong tubular protusions (FIG. 4A). In parallel, macrophages stablytransduced with a lentiviral vector containing the human mitochondrialfrataxin tagged with GFP (LV-hFXN-GFP) were used. Mitochondria were thenlabeled with red MitoTracker in the co-culture assay. Transfer ofhFXN-GFP-bearing mitochondria via TNTs was observed from the macrophagesto the diseased fibroblasts (FIG. 4B). Together, these resultsdemonstrate the ability of macrophages to transfer frataxin-bearingmitochondria to FRDA cells via TNTs, suggesting a potential mechanism ofrescue by HSPC-derived cells in the YG8R model.

HSPC-Derived Microglial Cells/Macrophages Enable Neuronal and MuscularCross-Correction In Vivo.

To assess whether transfer of mitochondrial proteins occurs in vivo,YG8R mice were transplanted with HPSCs isolated from DsRed Cox8-GFPmice. Cox8-GFP punctae were detected within the DsRed-expressingmicroglial cells but also within neurons in brain, spinal cord and DRGs(FIGS. 4C and 8). It was observed that neurons containing Cox8-GFP werein contact with one or more DsRed⁺ microglial branch extensions (FIG.4C) and GFP⁺ punctae were also observed within these microglialprocesses (FIG. 4D). These data suggest the involvement of themicroglial membrane projections in the transfer of Cox8-GFP proteinsfrom HSPC-derived microglia to host neurons. Quantification in spinalcord tissue revealed that about 50% of neurons contained Cox8-GFP (FIGS.4E and 9A-9D). Cross-correction of frataxin from microglia to neuronswas also demonstrated by transplanting YG8R mice with HSPCs isolatedfrom DsRed-transgenic mice and stably transduced with LV-hFXN-GFP (FIG.4F). In addition, evidence of transfer was apparent in heart andskeletal muscle, in which Cox8-GFP was detected in host cardiac/muscularmyocytes in apposition to graft-derived macrophages (FIG. 8). Together,these results represent the first demonstration of mitochondrial proteintransfer from microglia to neuronal cells and provide strong indicationthat cross-correction is involved in HSPC-mediated rescue of FRDAphenotype in this animal model.

pCCL-FXN Constructs and In Vitro Testing.

For developing a HSC gene therapy approach for FRDA, pCCL-EFS-X-WPRE(pCCL) LV were used. This vector backbone is the one used for the futureclinical trial for cystinosis. A central polypurine tract (cPPT)fragment that increases the nuclear import of viral DNA was added to theCCL vector backbone. A Woodchuck hepatitis virus PosttranslationalRegulatory Element (WPRE) is present to boost titer and gene expression.However, its open-reading frame was eliminated because it overlappedwith the woodchuck hepatitis virus X protein, a transcriptionalactivator involved in the development of liver tumors. Transgeneexpression is driven by the ubiquitously expressed short intron-lesshuman Elongation Factor 1 alpha promoter (EFS, 242 bp). The human FXNcDNA (633 bp), corresponding to the canonical frataxin (isoform I, FXNI) found in mitochondria, was amplified by PCR and inserted into pCCLgenerating pCCL-EFS-hFXN (FIG. 5A), and upstream eGFP generatingpCCL-EFS-hFXNeGFP. Additionally, a lentviral construct that carries Cas9enzyme and guide RNA was generated to remove the expansion of GAArepeats in the first intron of frataxin gene. The integrity of theconstructs was verified by sequencing and restriction enzyme digestion.LV virus particles were produced and titered as previously described.

YG8R fibroblasts were transduced with pCCL-EFShFXNeGFP, resulting in˜100% GFP⁺ cells, which were tested for their functional rescue. It wasreported that frataxin deficiency results in increased cellsusceptibility to H₂O₂ toxicity. Compared to WT fibroblasts, significantreduction in cell survival after exposure to H₂O₂ was observed in YG8Rfibroblasts. Improved survival was demonstrated in theFXN-GFP-transduced fibroblasts compared to YG8R controls but did notreach the WT level (FIG. 5B).

The data provided herein demonstrates that neurological and muscularpathology can be fully prevented in the YG8R mice transplanted with WTHSPCs at 2 months of age. Finally, the data suggests that the mechanisminvolved in this rescue is the transfer of frataxin-bearing mitochondriafrom the HSPC-derived phagocytic cells to the diseased cells via TNTs.

Example 2 Materials and Methods

Animals. YG8R mice with a deletion of murine Fxn gene (mFxn) andexpressing mutant human FXN gene (hFXN) containing 190+90 GAA repeatexpansion were generated in a C57BL/6J background as previouslydescribed (Al-Mahdawi, et al., GAA repeat instability in Friedreichataxia YAC transgenic mice. Genomics 84, 301-310 (2004); Al-Mahdawi, etal., GAA repeat expansion mutation mouse models of Friedreich ataxiaexhibit oxidative stress leading to progressive neuronal and cardiacpathology. Genomics 88, 580-590 (2006), both of which are incorporatedherein by reference). Breeding pairs consisted of females heterozygousfor Fxn and males heterozygous for Fxn and hemizygous for FXN(B6.Cg-Fxntm1Mkn Tg(FXN)YG8Pook/J), and were purchased from JacksonLaboratory (Bar Harbor, Me.). YG8R mice and wild-type (WT) mice used ascontrols for these studies were obtained from these breeders. Genotypingwas performed using the following primers:

(SEQ ID NO: 5) mfxn-F: 5′-CTTCCCTCTACCCTGCCTTC-3′ (SEQ ID NO: 6)mfxn-R: 5′-GGAGAACAGTGGACACAGTAACA-3′ (SEQ ID NO: 7)PGK-NEO: 5′-CATCGCCTTCTATCGCCTTCT-3′ (SEQ ID NO: 8)FXN-F: 5′-GGGCAGATAAAGGAAGGAGATAC-3′ (SEQ ID NO: 9)FXN-R: 5′-ACGATAGGGCAACACCAATAA-3′.

Transgenic mice constitutively expressing GFP(C57BL/6-Tg(ACTB-EGFP)1Osb/J) or DsRed (B6.Cg-Tg(CAG-DsRed*MST) Nagy/J)were also purchased from Jackson Laboratory. The mtGFP-Tg transgenicmice (C57BL/6J-Tg(CAG-Cox8/EGFP)49Rin) expressing the Cox8-GFPmitochondrial fusion protein were purchased from the RIKEN BioResourceCenter through the National Bio-Resource Project of the MEXT (Wako,Saitama, Japan). mtGFP-Tg mice were backcrossed with Dsred-Tg mice toproduce DsRed-mtGFP-tg mice. Genotyping for mt-GFP was done by PCR aspreviously described (Shitara, et al., Non-invasive visualization ofsperm mitochondria behavior in transgenic mice with introduced greenfluorescent protein (GFP). FEBS Lett 500, 7-11 (2001)). Mice weremaintained in a temperature- and humidity-controlled animal facility,with a 12-h light-dark cycle and free access to water and food. Bothmale and female mice were used in all experiments.

Frataxin-GFP Lentivirus Construction, Production and Titer.

The Self Inactivated (SIN)-lentivirus vector (LV), pCCL-EFS-X-WPRE-GFP(pCCL-GFP) was used for stable gene transfer in HSPCs and macrophages.The vector backbone contains the intron-less human elongation factor 1apromoter to drive transgene expression. The human FXN cDNA (Clone ID5300379, GE Healthcare; 633 bp) corresponding to the canonical frataxin(isoform I, FXN I) found in mitochondria (Perez-Luz, et al., Delivery ofthe 135 kb human frataxin genomic DNA locus gives rise to differentfrataxin isoforms. Genomics 106, 76-82 (2015), incorporated herein byreference) was amplified by PCR using the following primers: F:5′-TTAGGATCCATGTGGACTCTCG-3′ (SEQ ID NO: 10) and R:5′-AGAGGATCCAGCATCTTTTCCG-3′ (SEQ ID NO: 11); and inserted into pCCL atthe BamH1 restriction site in phase with the GFP cDNA. LV were producedand titered as previously described (Harrison, et al., Hematopoieticstem cell gene therapy for the multisystemic lysosomal storage disordercystinosis. Mol Ther 21, 433-444 (2013), incorporated herein byreference).

Bone Marrow Cell Isolation, Transduction Transplantation and EngraftmentDetermination.

Bone marrow cells were flushed from the femurs of 6-8 week old YG8Rmice, GFP transgenic mice, DsRed transgenic mice or DsRed mt-GFPtransgenic mice. Hematopoietic stem and progenitor cells (HSPCs) wereisolated by immunomagnetic separation using anti-Sca1 antibodyconjugated to magnetic beads (Miltenyl Biotec, Auburn, Calif.). Sca1⁺cells were directly transplanted by tail vein injection of 1×10⁶ cellsre-suspended in 100 μl of PBS into lethally irradiated (7Gy; X-Rad 320,PXi) YG8R mice. Prior to transplantation, Sca1⁺ cells from the DsRedtransgenic mice were first transduced with LV-hFXN-GFP using amultiplicity of infection (MOI) of 10 in presence of polybrene (4 mg/mL)in retronectin-coated (20 g/mL) 24-well plates at a density of 2×10⁶cells per well for 16 hours in StemSpan medium (StemCell Technologies)supplemented with SCF, TPO, FLT3 ligand (100 ng/mL each), and IL6 (20ng/mL) cytokines (PeproTech). Bone marrow cell engraftment of thetransplanted cells was measured in peripheral blood 2 monthspost-transplantation; blood samples freshly harvested from the tailswere treated with red blood cell lysis buffer (eBioscience, San Diego,Calif.) and subsequently analyzed by flow cytometry (BD Accuri C6, BDBiosciences) to determine the proportion of GFP- or DsRed-expressingcells.

Behavioral tests. WT mice, YG8R mice, YG8R mice transplanted withmfxn^(−/−) hFXN⁺ HSPCs, and YG8R mice transplanted with either WT GFP orDsRed/mt-GFP HSPCs were tested at both 5 and 9 months of age beforebeing sacrificed for tissue analysis. Rotarod analysis was performedusing a Roto-rod Series 8 apparatus (Ugo Basille, Comerio, Italy). Therod was a knurled plastic dowel (6.0 cm diameter) set at a height of 30cm. During training the mice were placed on the stationary rotarod for30 sec before the trial was initiated. Then each mouse was given 4trials per day, with a 60 sec inter-trial interval on the acceleratingrotarod (4-40 rpm over 5 min). The latency to fall was recorded for eachtrial. Locomotor activity was measured using an automated monitoringsystem (Kinder Associates, San Diego, Calif.). Polycarbonate cages(42×22×20 cm) containing a thin layer of bedding material were placedinto frames (25.5×47 cm) mounted with photocell beams. Each mouse wasplaced into the open field and all movements were recorded over a60-second testing period. Grip strength was measured using a deviceconsisting of a 10 cm long T-shaped bar connected to a digitaldynamometer (Ugo Basile, Comerio, Italy). Animals were held by the tailand placed before the bar, allowed to grip the bar with their forelimbs,and then gently pulled backwards until the bar was released. Tenconsecutive measurements were made for each animal and both the averageand maximal readouts were recorded. Gait measure (stride length) wascollected using an automated gait analysis system (CatWalk (NoldusInstruments)). Animals were placed at one end of the walkway and allowedto run down the length of the walkway, as two light sources illuminatedthe surface contact of paws with the glass floor, producing an image ofa paw print. During locomotion, the glass walkway was filmed from belowby a video camera. The CatWalk software program was used to analyzerecorded footage, define individual paw prints (e.g., left forepaw,right hindpaw), and give readouts of multiple parameters of gait.Testing was administered daily for 5 days. Only unbroken bouts oflocomotion, during which animals ran down the walkway at a consistentspeed, were used for analysis.

Primary Fibroblast and Macrophage Isolation, and Transduction.

Fibroblasts were generated from skin biopsies of neonate of YG8R mice.Cultures were maintained using high-glucose DMEM (Dulbecco's modifiedEagle's medium; Life Technologies, Carlsbad, Calif.) supplemented with10% fetal bovine serum (FBS; Gibco, Life Technologies) and 1%penicillin/streptomycin (PenStrep; Gibco) at 37° C. under 5% CO₂.Primary macrophages from DsRed mt-GFP mice were derived from bone marrowcells. Bone marrow cells were flushed from the femurs of 6-8 week oldmice and kept in culture in RPMI medium with 10% FBS, 1% PenStrep and10% L929 conditioned medium 29 at 37° C. under 5% CO². For macrophagetransduction with pCCL-FXN-eGFP, the IC-21 macrophage cell line was used(American Type Culture Collection, catalog #TIB-186) and cultured inRPMI 1640 medium (Gibco). Six-well plates were coated with retronectin(20 μl/ml; Takara Bio) following the manufacturer's instructions. IC-21macrophages were plated at 250,000 cells in 2 ml per well and transducedwith pCCLFXN-eGFP using a MOI of 15. Media was changed 24 hours aftertransduction.

Live Imaging.

YG8R fibroblasts were co-cultured with DsRed Cox8-GFP or macrophagesstably transduced with a lentivirus expressing hFXN-GFP as previouslydescribed (Naphade, et al., Brief reports: lysosomal cross-correction byhematopoietic stem cell-derived macrophages via tunneling nanotubes.Stem Cells 33, 301-309 (2015), incorporated herein by reference).Briefly, 75,000 fibroblasts were co-cultured with equal number ofmacrophages in glass-bottomed culture dishes (MatTek Corp, Ashland,Mass.). hFXN-GFP co-cultures were stained with 50 nM MitoTracker(Invitrogen) for 45 minutes prior to imaging. Confocal live imaging wasperformed 1 and 2 days later using Perkin Elmer UltraView Vox SpinningDisk Confocal with ×40 (Numerical aperture (NA)=1.30) and ×60 (NA=1.42)oil objective at 37° C. under 5% CO₂. Images were captured, processed,and analyzed using Volocity Software (Perkin Elmer, Waltham, Mass.).

Mouse Frataxin Quantitative RT-PCR.

Total RNA was prepared from snap-frozen skeletal muscle, brain and heartbiopsies using the RNeasy Lipid and Fibrous Tissue kits (Qiagen)according to manufacturer's instructions. cDNA was then prepared usingiScript cDNA Synthesis kit (Bio-Rad). Commercial TaqMan probes specificto mouse frataxin were employed to quantitate expression (AppliedBiosystems).

Oxidative Stress Detection.

Protein lysates from tissues directly snap-frozen in liquid nitrogenafter dissection were prepared using RIPA buffer (Sigma) containingproteases inhibitors (Roche) as previously described (Campuzano, et al.,Frataxin is reduced in Friedreich ataxia patients and is associated withmitochondrial membranes. Human molecular genetics 6, 1771-1780 (1997),incorporated herein by reference). For each assay, μg of protein wasused after total protein concentration was determined using the BCAassay. Proteins were then derivatized by adding1×2,4-Dinitrophenylhydrazine (DNPH) solution contained in the OxyBlotProtein Oxidation Detection kit (Chemicon International) according tomanufacturer's instructions. Samples were applied to electrophoresis andtransferred to a PVDF membrane. After blocking with 1% BSA/PBS-T,membrane was incubated with Rabbit anti-Dinitrophenyl (DNP) antibodyfollowed by a Goat anti-rabbit HRP conjugate, and visualized using ECLkit (Pierce). Protein levels were normalized using an anti-Tubulin(ab6161, Abcam) antibody and band intensity was quantified usingImagePro software (Media Cybernetics).

Mouse Mitochondria RT2 Profiler PCR Array. RNA was isolated from thecerebrum using the RNeasy Lipid Tissue Mini Kit (Qiagen) and 0.5 μg wasthen reverse transcribed with the iScript cDNA Synthesis Kit (Bio-rad).Samples were mixed with SYBR green and equally loaded into all wells ofthe Mouse Mitochondria RT2 Profiler PCR Array (Qiagen, Cat. no.PAMM-087Z) and amplified per manufacture's recommendation on the CFX96Thermocycler (Bio-rad). Ct data was exported and fold change calculatedusing the delta Ct method between sample genes and a panel ofhousekeeping controls.

Lactate/Pyruvate Analysis.

Muscle biopsies (10 mg) were homogenized in ice in 1 ml of ice cold 40%acetonitrile (containing 0.1% formic acid)/40% methanol/20% H₂O) using atissue grinder (dounce), followed by centrifugation for 10 minutes at13,000×g. The extraction solution contained stable isotope of lactate(¹³C3 sodium-lactate, Cambridge Isotope Laboratories, Inc.).Supernatants were removed, dried in a speed vac/lyophilizer system, andre-suspended in 150 μl 0.1% formic acid. Pellets were re-dissolved in0.1N NAOH and protein content measured using a bicinchoninic acid (BCAassay). 5 μl of each resuspended supernatant was injected on a C18-pfpHPLC column (Mac-Mode Analytical, Chadds Ford, Pa.), as previouslydescribed (Gertsman, et al., Validation of a dual LC-HRMS platform forclinical metabolic diagnosis in serum, bridging quantitative analysisand untargeted metabolomics. Metabolomics 10, 312-323 (2014),incorporated herein by reference), and coupled to an API-4000 triplequadrupole mass spectrometer (AB Sciex). MRM (molecular reactionmonitoring) for lactate (89>43), ¹³C3-lactate (92>45), and pyruvate(87>43 and 87>87) were used during the acquisition. Lactate and pyruvatepeaks were both normalized to ¹³C3 lactate. Both lactate and pyruvatewere further normalized to protein content (mg) prior to calculation ofthe final lactate/pyruvate (L/P) peak area ratios used in FIG. 3B. Sincethe ratio is expressed in terms of normalized peak areas, the ratiovalues should not be confused with those determined from absoluteconcentration measurements as performed in previous studies measuringL/P, but still effective for examining relative differences betweencohorts.

Vacuole Imaging and Quantification.

Dorsal root ganglia (DRG) from lumbar level 5 (L5) were collected,sectioned at 30 m intervals using a cryostat, and mounted ongelatin-coated slides. DRG sections were stained with thionin (Nisslstain) for visualization of neuronal cell bodies. Three DRGs per subjectwere acquired at 60× magnification using a BZ-X700 fluorescentmicroscope (Keyence). The presence of vacuoles in each DRG was tracedand measured by a blinded experimenter in duplicate using ImageJ;vacuoles were defined as extremely circular white (Nissl negative) areaswith smooth edges within DRG neurons. Number of vacuoles and area ofvacuolar space relative to entire area of each DRG section was comparedacross genotypes.

Heart histology and iron quantification. For histological preparations,terminally anesthetized mice were fixed by intracardial perfusion with10% formalin. Fixed tissues were dissected, embedded in paraffin wax,and sectioned by standard methods. Sections were deparaffinized andstained using Perl's technique to detect ferric iron as previouslydescribed (Al-Mahdawi, et al., GAA repeat expansion mutation mousemodels of Friedreich ataxia exhibit oxidative stress leading toprogressive neuronal and cardiac pathology. Genomics 88, 580-590(2006)). Whole heart sections were imaged on the Keyence FluorescenceMicroscope and a single wide-field image stitched together. UsingImagePro Preimier Software (MediaCybemetics), levels of iron stainingwere assessed by isolating the blue channel, measuring the area ofsignal and then dividing from total area of the section. Values werereported normalized to wild-type levels.

Immunofluorescence and Image Acquisition.

Heart and muscle tissues were fixed in 5% paraformaldehyde, equilibratedin 20% sucrose overnight and frozen in Tissue-Tek Optimal CuttingTemperature (OCT) medium at −80° C. (Sakura Finetek U.S.A, Torrance,Calif.); 10 μm sections were cut. DRG, brain, and spinal cord tissuewere fixed in paraformaldehyde, cryopreserved in 30% sucrose, and frozenin OCT medium. For DRGs, tissue was cut into 20 μm sections and directlymounted to gelatin-coated slides. For brain and spinal cord, tissue wassectioned to 30 μm and collected as free-floating sections. Forimmunofluorescence, tissues were incubated with the followingantibodies: rat anti-CD68 (1:100; BioLegend 137001), Biotin ratanti-MHCII (1:100; BD Pharmigen 553622), rabbit anti-GFP (1:500; Abcamab290), chicken anti-GFP (1:1500, Abcam ab13970), rabbit anti-Iba1(1:1500; Wako #019-19741), goat anti-mCherry (1:1000, Sicgen AB0040),mouse anti-NeuN (1:500; Millipore MAB377), rabbit anti-MBP (1:200,Millipore AB980), mouse anti-NF200 (1:500, Millipore MAB5262), mouseanti-α-Actinin (1:400; Sigma), Rabbit anti-von Willibrand factor (1:300;Chemicon), DAPI (1:500; Molecular Probes), Bodipy-Phalloidin (1:100;Molecular Probes). The appropriate AlexaFluor-conjugated secondaryantibodies (Invitrogen) were used for visualization of antigens. Imageswere acquired using the LSM 880 with Airyscan confocal microscope(Zeiss), a Keyence BZ-X710 digital microscope system for high resolutionstitching images of tissue sections, or an Olympus FV1000 confocalmicroscope for live imaging. Confocal image stacks were analyzed withIMARIS Software (Bitplane, Oxford Instruments).

Quantification of Neuronal Cross-Correction.

The entire gray matter region of lumbar spinal cord sections from threeYG8R mice transplanted with Cox8-GFP HSPCs and an untransplanted controlwere stained with NeuN and imaged at 20× on the LSM 880 confocalmicroscope (Zeiss). NeuN+ neuronal cells were outlined and counted usingImagePro Plus software (Media Cybernetics) and then assessed for GFPpositivity which was reported as a percentage of total NeuN cells (FIG.8). All acquisition, filtration and processing steps were performedidentically on the GFP channel between all samples.

Clearing of Mouse Spinal Cord.

A 6-mm segment of cervical spinal cord from a mouse at 3 monthspost-transplantation with DsRed⁺ HSPCs was processed for opticalclearing as previously described (Chung, et al., Structural andmolecular interrogation of intact biological systems. Nature 497,332-337 (2013), incorporated herein by reference). Briefly, PFA-fixedtissue was infused with hydrogel monomer solution (4% PFA, 4%acrylamide, 0.05% bis-acrylamide) and thermally polymerized. Lipids werethen passively extracted in SDS-containing borate buffer at 37° C. for 4weeks, until tissue was cleared. Clarified tissue was incubated inRapidclear CS for 1 day and mounted using a Wilco dish. Tissues werethen imaged using an Olympus FV1200 system equipped with a 10×water-immersion objective (numerical aperture: 0.6; working distance: 3mm; stack size: 1.65 mm; step size, 5 μm).

Statistics.

No animals were excluded from the experiments. Experimenters wereblinded to the genotype of the specific sample to every extent possible.Power calculation analysis was not performed. All data displayed normalvariance except DRG vacuole measurements. For normal data andmitochondrial PCR array data, one-way analysis of variance (ANOVA) wasperformed, followed by post-hoc Student's t-test to determinestatistical significance using GraphPad Prism 7.01 (GraphPad Software,La Jolla, Calif.). Oxidative stress measurements employed one-tailedt-tests with the assumption that YG8R oxidation levels would be higher.For vacuole measurements, the Mann-Whitney nonparametric test correctedfor multiple testing by the Bonferroni correction was used. In vitroexperiments were performed in biological triplicates. Error bars denotes.e.m. The level of significance is indicated as follows: *P<0.05,**P<0.01, ***P<0.005.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method of treating a mitochondrial disease or disorder in a subjectcomprising: introducing a functional human gene associated with amitochondrial disease or disorder into hematopoietic stem and progenitorcells (HSPCs) of the subject; and transplanting the HSPCs into thesubject, thereby treating the mitochondrial disease or disorder.
 2. Themethod of claim 1, wherein the step of introducing comprises contactinga vector comprising a polynucleotide encoding the functional human geneand a promoter with the HSPCs or by using a gene editing approach andallowing expression of hFXN.
 3. The method of claim 1, wherein themitochondrial disease or disorder is selected from the group consistingof mitochondrial myopathy, diabetes, Leigh syndrome, Neuropathy ataxia,retinitis pigmentosa, NARP syndrome, MNGIE, MERRF, MELAS, and FRDA. 4.The method of claim 1, wherein the subject is a mammal.
 5. The method ofclaim 4, wherein the subject is human.
 6. The method of claim 1, whereinthe vector is a viral vector selected from the group consisting of alentiviral, adenoviral, and AAV vector.
 7. (canceled)
 8. The method ofclaim 1, wherein the transplantation of the HSPCs into the subjectcorrects neurologic, cardiac and muscular complications within about6-12 months post-transplantation.
 9. The method of claim 1, wherein thestep of introducing the functional human gene into the HSPCs isperformed ex vivo.
 10. A vector comprising a promoter functionallylinked to a polynucleotide encoding hFXN.
 11. The vector of claim 10,wherein the vector is a viral vector selected from the group consistingof a lentiviral, adenoviral, and AAV vector.
 12. The vector of claim 11,wherein the vector is pCCL-FRDAp-FXN or pCCL-EFS-FXN.
 13. An isolatedmammalian host cell containing the expression vector according to claim10, wherein the cell is an HSPC.
 14. (canceled)
 15. A method of treatinga mitochondrial disease or disorder in a subject comprising contactingcells expressing hFXN from the subject with a vector encoding a geneediting system that when transfected into the cells removes atrinucleotide extension mutation of endogenous hFXN, thereby treatingthe mitochondrial disease or disorder.
 16. The method of claim 15,wherein the gene editing system is selected from the group consisting ofCRISPR/Cas, zinc finger nucleases, and transcription activator-lifeeffector nucleases.
 17. The method of claim 15, wherein the step ofcontacting comprises administering to the subject an effective amount ofthe vector.
 18. The method of claim 15, wherein the step of contactingcomprises obtaining a sample of cells from the subject, transfecting thegene editing system into the sample of cells, and thereafter,transplanting the transfected cells into the subject.
 19. The method ofclaim 18, wherein the sample of cells is selected from the groupconsisting of blood cells and HSPCs.
 20. The method of claim 2, whereinthe gene editing technology is CRISPR/Cas9.
 21. The method of claim 1,wherein the functional gene is frataxin (hFXN).
 22. The method of claim21, wherein the mitochondrial disease is FRDA.